# 1. Introduction

A permanent tooth that erupts into the mouth does not yet have a finished root. After eruption, root formation continues for roughly two to three more years, during which the dentinal walls thicken, the root lengthens and the apical foramen narrows to a mature size. When pulp disease or trauma interrupts this process, the clinician is left with a tooth whose root is short, whose walls are thin and whose apex is wide open. This is the immature tooth with an open apex, and managing it is one of the more demanding problems in paediatric and adolescent endodontics.

The difficulty is both mechanical and biological. Mechanically, a divergent or "blunderbuss" canal has no natural apical constriction against which a gutta-percha cone can be compacted, so conventional obturation risks extrusion of sealer and filling material into the periapical tissues. The thin walls are also prone to cervical fracture, particularly after chemical or mechanical weakening. Biologically, loss of the pulp before root completion means that the tooth stops forming dentin, stays structurally weak and cannot be given the thick, buttressed root of a mature tooth. The practical question therefore changes from "how do I seal this canal?" to one of three alternatives: how can the vital pulp be kept so the root completes by itself (apexogenesis), how can a hard tissue barrier be induced or created at the apex (apexification), or how can a living tissue be re-established inside the canal so that the root continues to develop (regenerative endodontic procedures)?

This seminar covers the biology of the immature apex, diagnosis and case selection, and each treatment family with its technique, materials, evidence and limitations. It ends with a comparison of materials, emerging approaches, special situations, and a summary of outcomes.

## 1.1 Terminology

The literature uses several terms loosely, so definitions help.

- **Immature tooth / open apex:** a permanent tooth in which root development is incomplete, usually with an apical diameter wider than the canal diameter at the mid-root, and walls that are thin and may be divergent at the apex.
- **Apexogenesis:** a vital pulp therapy that maintains the health of the radicular pulp so that physiological development and formation of the root end continue. It requires vital radicular pulp tissue.
- **Apexification:** a method of inducing a calcified barrier in a root with an open apex or the continued apical development of an incompletely formed root in teeth with necrotic pulp. The barrier may be biologically induced (calcium hydroxide) or artificial (an MTA or bioceramic plug).
- **Regenerative endodontic procedure (REP):** biologically based procedures designed to replace damaged structures, including dentin and root structures and cells of the pulp-dentin complex. Older labels include revascularization, revitalisation and "maturogenesis". These are not synonyms, but the terms overlap in practice and in many papers.
- **Pulp necrosis with apical periodontitis:** the usual starting point for apexification or REP.

## 1.2 Epidemiological and clinical context

Most open-apex teeth that need endodontic treatment are maxillary incisors, injured between about 7 and 11 years of age. Dental trauma is the leading cause, and complicated crown fractures, luxation injuries and avulsion with delayed replantation all contribute. Other causes include deep caries in immature first permanent molars and premolars, and developmental anomalies such as dens invaginatus and dens evaginatus, where a fine pulp horn is exposed through occlusal wear or fracture of the tubercle before the root completes. The relevance is practical: the same tooth will be kept in the mouth of the patient for seven or eight decades, so a treatment that weakens the root, or that accepts a high risk of cervical fracture, has long-term costs.

# 2. Biology of the Immature Apex

## 2.1 Stages of root development

Several systems describe how far a root has developed. Two are used in treatment planning.

**Nolla stages (1 to 10).** Nolla's scheme describes tooth calcification: stage 6 is crown completion, stage 7 is one third of the root formed, stage 8 is two thirds of the root formed, stage 9 is root nearly complete with an open apex, and stage 10 is apical closure. Teeth at stages 7 to 9 are those usually considered for the techniques in this seminar.

**Cvek's stages of root development.** Cvek (1992) classified the root of an incisor into five stages: stage 1 where root length is less than half of its final length with an open apex; stage 2 where the root is about half formed; stage 3 about two thirds formed with an open apex; stage 4 almost full length with an open apex; stage 5 full length with a closed apex. The classification is useful because the risk of cervical fracture after prolonged calcium hydroxide therapy tracks it closely: the less developed the root, the greater the risk of fracture.

Approximate eruption and root completion timelines in the permanent dentition help the clinician decide whether a tooth is likely to be immature: maxillary central incisors erupt at 7 to 8 years and complete roots at about 10; lateral incisors erupt at 8 to 9 and complete at about 11; premolars erupt at 10 to 12 and complete at about 12 to 14; first molars erupt at 6 and complete at about 9 to 10; second molars erupt at 12 and complete at 14 to 16. These ages are averages and vary by population and sex.

## 2.2 Histology of the developing root

Root formation is directed by Hertwig's epithelial root sheath (HERS), a double layer of epithelium that grows apically from the cervical loop of the enamel organ. HERS induces the adjacent dental papilla cells to differentiate into odontoblasts, which lay down root dentin. As the root lengthens, the sheath breaks up into the epithelial cell rests of Malassez, and cells from the dental follicle contact the dentin surface and form cementum. At the apex, HERS bends to form the epithelial diaphragm, which defines the apical foramen as it narrows.

Two tissue compartments at the apex matter for treatment: the **dental papilla**, which becomes the pulp, and the **apical papilla**, a loosely attached tissue at the root tip, continuous with the pulp but histologically distinct, with a cell-poor zone between them. The apical papilla is rich in mesenchymal stem cells. Sonoyama and colleagues described these stem cells from the apical papilla (SCAP) and showed that they are the likely source of primary odontoblasts that form root dentin (Sonoyama et al., 2008). Because the apical papilla receives its blood supply from the periapical tissues, rather than only from the pulp, it can survive pulp necrosis. This is the biological premise on which REPs rest: even when the canal space is infected and the pulp has died, SCAP and HERS may still be alive at the apex and able to respond when the canal is disinfected and a scaffold is made available.

## 2.3 Structural features that complicate treatment

- **Thin dentinal walls.** The canal is wide and the walls thin, so the root resists less occlusal and traumatic load. Aggressive instrumentation will weaken it further, which is why mechanical instrumentation is kept to a minimum or avoided in these cases.
- **Divergent apex.** The absence of an apical constriction prevents compaction and creates the risk of overfilling.
- **Short roots.** Crown to root ratio is unfavourable, and any further loss of root length (for example, from resorption) is poorly tolerated.
- **Large, vital-appearing periapical radiolucency.** In an immature tooth the apical papilla and dental follicle appear as a radiolucency around the apex, which may be mistaken for a lesion.
- **Difficult working length.** Electronic apex locators are less accurate with a wide open apex, so radiographic confirmation is needed.
- **Immature dentin and a rich blood supply** also explain why the pulp of young teeth has greater healing potential, which is the basis for vital pulp therapy being a strong first choice when the pulp is still alive.

# 3. Etiology

## 3.1 Dental trauma

Trauma is the major cause of pulp necrosis in immature incisors. Mechanisms include:

- **Complicated crown fracture** with pulp exposure, which allows bacterial invasion.
- **Luxation injuries** (lateral luxation, intrusion and extrusion). These sever or stretch the apical neurovascular bundle. The wide apex gives some chance of pulp revascularisation, particularly in teeth with an open apex of more than about 1 mm (Andreasen's work on luxation injuries), but intrusion and severe lateral luxation have a high rate of necrosis.
- **Avulsion** with replantation after extended dry time. Revascularisation of the pulp is feasible with immediate replantation of immature teeth, but delayed replantation almost always leads to necrosis and often to inflammatory root resorption.
- **Concussion and subluxation**, which produce a smaller risk of necrosis but still need monitoring.

## 3.2 Caries and restorations

Deep caries in newly erupted first molars and premolars can cause pulp exposure before root completion. These are often managed with vital pulp therapy, because the inflammation is frequently confined to the coronal pulp.

## 3.3 Developmental anomalies

- **Dens evaginatus** (central tubercle on premolars, common in Asian and Indigenous American populations). The tubercle contains a pulp horn. Occlusal wear or fracture of the tubercle exposes it, and a premolar may undergo necrosis before root completion.
- **Dens invaginatus** creates a deep infolding with a thin enamel lining that readily allows bacterial access to the pulp.
- **Others**: enamel hypoplasia, taurodontism and fusion or gemination can also lead to early pulpal compromise.

## 3.4 Iatrogenic and other causes

Deep cavity preparations, thermal injury, orthodontic movement and periodontal-endodontic lesions are less frequent causes. Long-standing, untreated pulpitis and periapical infection in the child are also found when dental care is delayed.

# 4. Diagnosis

## 4.1 History and clinical examination

The history should establish the date and nature of trauma, any previous treatment, and the symptoms (spontaneous pain, swelling, a sinus tract). Examination includes percussion and palpation, mobility, probing depths (to rule out vertical root fracture or a periodontal defect), the presence of a sinus tract (with gutta-percha tracing), and crown discolouration. A grey crown suggests necrosis or haemorrhagic breakdown products.

## 4.2 Pulp sensibility testing in immature teeth

Pulp tests are less reliable in immature teeth. Electric pulp testing and thermal tests depend on myelinated A-delta fibres, which mature later, and on the Raschkow plexus of nerve fibres under the odontoblast layer, which is incomplete in young teeth. Studies of traumatised teeth have found that false negative responses are common soon after injury and that sensibility may return months later as the neural supply recovers. Practical points:

- Test the contralateral tooth first so that the child knows what to expect.
- Use cold (refrigerant spray or ice) in preference to heat, and electric tests as a supplement.
- Interpret a single negative result with caution, and repeat the test at several visits.
- Consider laser Doppler flowmetry and pulse oximetry when available, since they assess blood flow rather than nerve response. They are mainly research tools, and clinical access is limited.
- Look at the whole picture: crown colour, radiographic periapical change, sinus tract, and failure of root development compared with the contralateral tooth.

## 4.3 Radiographic examination

A periapical radiograph, taken with a paralleling technique, is the first choice. Findings to look for include root length, wall thickness, width of the apex, periapical radiolucency, root resorption (surface, inflammatory or replacement), and the continuity of the lamina dura. In an immature tooth, a radiolucency of regular outline, with an intact lamina dura continuing around the follicle, and symmetrical with the contralateral tooth, is probably normal apical papilla and follicle. A radiolucency which is asymmetrical, with a loss of the lamina dura and clinical signs, indicates apical periodontitis.

**CBCT** is not routine. For small field of view, with a low dose, it is justified when planar films cannot answer the question: for example, in suspected root fracture, internal or external resorption, complex dens invaginatus, or when a surgical option is being considered. The ALARA principle applies, particularly in children.

Radiographic measurements useful for planning: the width of the apex (some authors cite an apical diameter of around 1 mm or more as helpful for REP success because it allows cell influx and revascularisation), the length of the root, the thickness of the walls at the cervical level, and the position of the cementoenamel junction (CEJ), because it is used as a reference level for placing the coronal barrier.

## 4.4 Diagnostic terms

The American Association of Endodontists terminology is commonly used: pulpal diagnoses include normal pulp, reversible pulpitis, symptomatic irreversible pulpitis, asymptomatic irreversible pulpitis, pulp necrosis and previously treated or initiated therapy. Periapical diagnoses are normal apical tissues, symptomatic apical periodontitis, asymptomatic apical periodontitis, acute apical abscess and chronic apical abscess. The choice of treatment in an immature tooth depends primarily on whether the pulp is vital or not, and on whether the pulp has any remaining healing potential.

# 5. Treatment Planning

## 5.1 The decision tree

The basic principle is to preserve the vital pulp wherever possible, because a living pulp finishes root formation better than any artificial method.

1. **Vital pulp, reversible or limited irreversible inflammation (including traumatic exposure):** vital pulp therapy. Indirect pulp capping, direct pulp capping, or partial (Cvek) pulpotomy, with full pulpotomy if the radicular pulp is inflamed in the coronal portion only. The goal is apexogenesis.
2. **Necrotic pulp, immature apex, and a restorable tooth in a cooperative patient:** a regenerative endodontic procedure is the first option in the AAE and ESE guidelines when the case is suitable, with MTA or bioceramic apexification as the alternative.
3. **Necrotic pulp where a post and core is needed, very short or very fractured roots, a non-cooperative patient, allergy to the medicaments:** apexification with a bioceramic apical plug and a bonded coronal restoration.
4. **Failure of non-surgical treatment:** surgical approach (apical surgery with retrograde MTA or bioceramic root-end filling), or extraction when the tooth is non-restorable.

## 5.2 Factors guiding the choice

The American Association of Endodontists case selection criteria for a regenerative procedure are: a tooth with a necrotic pulp and an immature apex; a pulp space not needed for a post or core for the final restoration; a compliant patient and parent; and a patient who is not allergic to the medicaments and antibiotics needed (ASA 1 or 2) (AAE, 2021). Informed consent should include the need for two or more appointments, use of antimicrobials, possible adverse effects (staining of crown or root, lack of response, pain or infection), and alternatives, which include MTA apexification, no treatment, or extraction.

Other factors worth weighing:

- **Stage of root development.** The less developed the root, the stronger the case for REP, because apexification leaves a short root with thin walls that is vulnerable to fracture. In a tooth with an almost complete root (Cvek stage 4), the biological benefit of REP is small and apexification may be satisfactory.
- **Presence of periapical infection.** Both techniques work in the presence of apical periodontitis, but REP depends on thorough disinfection since infection prevents regeneration.
- **Patient compliance.** REP requires two visits and a recall of at least 12 months, ideally longer.
- **Operator experience** and access to a microscope and the materials.
- **Restorative needs.** A tooth that needs intracanal retention for a core cannot be treated with REP, because the coronal canal must be sealed with a barrier and left.
- **Staining.** Anterior teeth are at risk of discolouration from minocycline and from certain MTA formulations; the aesthetic implications should be discussed with the family.
- **Allergies,** medical history, and the patient's age.

## 5.3 Informed consent in paediatric patients

The consent process is directed to the parent or guardian, with assent from the child. It should clearly state that the outcome of regenerative treatment is unpredictable, that root lengthening and wall thickening are not guaranteed, that the tooth may need further treatment, and that long-term follow-up is required.

# 6. Vital Pulp Therapy and Apexogenesis

## 6.1 Rationale

When the radicular pulp is vital, preserving it allows the odontoblasts and the apical papilla to continue root formation. The tooth ends up with a longer root, thicker walls and a normal crown to root ratio, and the long-term prognosis is far better than for a root that stopped developing. Vital pulp therapy (VPT) in immature teeth has been shown to be successful even when the pulp is exposed by trauma or caries, because the pulp of young teeth has a rich blood supply and a high capacity for healing. The AAE position statement on vital pulp therapy (2021) and the ESE position statement on deep caries and the exposed pulp (2019) both support VPT in appropriate cases.

## 6.2 Indications and contraindications

**Indications:**
- Traumatic pulp exposure in an immature permanent tooth (complicated crown fracture).
- Carious pulp exposure in an immature tooth with a vital pulp and a pulpal diagnosis of reversible pulpitis or, in current thinking, selected irreversible pulpitis where bleeding can be controlled.
- Developmental anomaly with minimal exposure and a vital pulp.

**Contraindications:**
- Spontaneous, lingering pain not relieved by analgesics suggesting extensive inflammation, in the older literature.
- Non-restorable tooth.
- Evidence of necrosis: sinus tract, swelling, periapical radiolucency in a tooth previously vital.
- Uncontrolled bleeding from the pulp after the pulpotomy.
- Internal resorption or calcification suggesting chronic pulp reaction.

## 6.3 Indirect pulp therapy (selective caries removal)

In deep caries where there is no exposure, selective caries removal to soft dentin on the pulpal floor in a deep lesion, with a hard-setting calcium hydroxide liner, glass ionomer, or bioceramic liner (such as Biodentine) and a sealed restoration, avoids pulp exposure. In an immature tooth this is preferred over complete caries excavation, which exposes the pulp in a large proportion of cases. The restoration must seal well, since the success of selective removal depends on bacterial starvation under an intact seal.

## 6.4 Direct pulp capping

A small mechanical or traumatic exposure is covered with a biocompatible material. Steps:

1. Anaesthesia and rubber dam isolation.
2. Disinfect the cavity and the exposure with 1.5% to 5.25% sodium hypochlorite on a cotton pellet or 2% chlorhexidine for 30 seconds to 1 minute (hypochlorite is also a haemostatic agent).
3. Bleeding should stop within about 5 minutes; if it does not, the inflammation is deeper and a pulpotomy is chosen.
4. Place the capping material directly on the exposure: MTA, Biodentine, or another calcium silicate cement. Calcium hydroxide (hard setting) is the historical material but suffers from tunnel defects in the dentinal bridge and slow dissolution under restorations.
5. Place a glass ionomer or resin-modified glass ionomer liner, and a final bonded restoration immediately to seal.

Compared with calcium hydroxide, MTA produces a thicker and more homogeneous dentin bridge, with less inflammation (as shown in comparative trials and meta-analyses), so calcium silicate cements are the current standard.

## 6.5 Partial pulpotomy (Cvek pulpotomy)

Cvek described this technique in 1978 for permanent incisors with complicated crown fractures. In his series, about 96% of 54 teeth followed for several years were successful, and the success was not significantly affected by the time elapsed since the injury or the size of the exposure.

**Technique:**
1. Anaesthesia and rubber dam. Disinfect the crown and the fracture surface.
2. Using a sterile, high-speed diamond bur with copious water coolant, remove the superficial pulp tissue to a depth of 2 to 3 mm below the exposure, or deeper until healthy, bleeding pulp is found. A sharp spoon excavator is less preferred since it can tear the pulp.
3. Irrigate with saline or sodium hypochlorite and achieve haemostasis by pressure with a cotton pellet moistened with 1.5% to 5.25% NaOCl for 1 to 5 minutes.
4. Cap the pulp with a 1.5 to 3 mm layer of MTA, Biodentine, or hard-setting calcium hydroxide, with MTA and Biodentine preferred.
5. Seal the cavity with a glass ionomer liner and the definitive bonded composite restoration, either the same visit or after confirming set.
6. Clinical and radiographic follow-up at 1, 3, 6 and 12 months, then yearly. The tooth should give a normal response to sensibility tests, continue root formation and have no periapical change.

If bleeding cannot be controlled within about 10 minutes, extend the pulpotomy deeper, ending in a full coronal pulpotomy.

## 6.6 Full (coronal) pulpotomy

The whole coronal pulp is amputated at the canal orifices, the radicular pulp stump is dressed with MTA or Biodentine, and the tooth is restored. It is used in immature molars with carious exposure and, increasingly, in permanent teeth with symptoms of irreversible pulpitis as a definitive treatment. The aim is to allow the roots to complete (apexogenesis). Success rates reported in systematic reviews for MTA and similar materials are high, above 90% in many series, although the quality of evidence is variable.

## 6.7 Outcomes and when VPT fails

Failure presents as pain, sinus tract, periapical radiolucency, internal resorption, pulp canal obliteration or arrested root development. After failure, the tooth is treated as a necrotic immature tooth by REP or apexification. A well-performed VPT does not prevent a subsequent REP or apexification, so there is little to lose by trying it in suitable cases.

# 7. Apexification

## 7.1 History and concept

Apexification became established in the 1960s with Kaiser (1964) and Frank (1966), who described the use of calcium hydroxide in the necrotic open-apex tooth to induce a calcified barrier at the apex. The term is now taken to cover both the traditional long-term (multi-visit) calcium hydroxide technique and the single- or two-visit technique using an artificial apical barrier. Neither technique encourages continued root development: the root remains short, and the walls remain thin. This is the main biological limitation of apexification compared with REP.

The objectives are:
- Eliminate infection from the root canal system.
- Establish an apical stop or barrier against which obturation can be placed.
- Restore the crown so that the tooth is functional and aesthetic.

## 7.2 Preliminary steps common to all apexification techniques

1. **Anaesthesia and isolation.** Local anaesthesia (with vasoconstrictor), rubber dam. In a young child, a split-dam technique or clamp placement on adjacent teeth is used.
2. **Access.** Conservative but sufficient access to give straight-line entry to the canal. Magnification is highly desirable.
3. **Working length.** Radiographic working length, since electronic apex locators can be unreliable in wide open apices; some operators use an apex locator and check radiographically. The working length is taken about 1 mm short of the radiographic apex.
4. **Cleaning.** Minimal or no filing; at most, gentle circumferential filing with a large file, to avoid weakening the walls. Cleaning relies on irrigation. Sodium hypochlorite (1% to 2.5%, in contrast to the 1.5% to 3% range for REP) is delivered with a side-vented or closed-end needle placed short of the apex, to avoid extrusion. Activation with ultrasonic or sonic devices (passive ultrasonic irrigation) and the EndoVac apical negative pressure system have been used to improve disinfection with a lower risk of extrusion. Chlorhexidine 2% can be used as a final rinse where tissue dissolution is not required, although it does not mix well with hypochlorite (a brown precipitate) so saline is used between solutions.
5. **Interappointment medicament.** Calcium hydroxide for 1 to 4 weeks to disinfect the canal before placing a barrier. Many clinicians still use this step before an MTA plug in cases with infection.

## 7.3 Long-term calcium hydroxide apexification

### Technique
1. After cleaning and irrigation, dry the canal with paper points and fill it with a calcium hydroxide paste to the working length. The paste is introduced with a Lentulo spiral, a syringe, or an injectable system, and condensed with pluggers or pre-fitted large paper points so that the whole canal is filled.
2. Seal the access cavity with a double seal (for example, a cotton pellet with glass ionomer or a temporary filling such as IRM or Cavit).
3. Review at 3 months. Take a radiograph and check for barrier formation. The paste is renewed every 3 months until a barrier is seen, or earlier if the paste washes out (as seen on a radiograph) or the tooth becomes symptomatic.
4. A barrier is tested by gently probing with a paper point or file. A "tactile" and radiographic assessment shows when a hard tissue has formed. This takes a mean of about 5 to 20 months, with a range up to 24 months or more depending on the study and the stage of root development.
5. When the barrier is judged complete, remove the paste by irrigation and use a gentle, circumferential action; obturate with warm gutta-percha (vertical compaction or thermoplasticised) and sealer, and restore the crown with a bonded restoration.

### Vehicles and additives
Calcium hydroxide has been mixed with sterile water, saline, local anaesthetic solution, methylcellulose, glycerine, propylene glycol, iodoform-based pastes (Vitapex, Metapex, which are pre-mixed silicone-oil/iodoform/calcium hydroxide), and with camphorated parachlorophenol (CMCP) in older reports. Pastes with radiopacifiers (barium sulfate) allow radiographic assessment. Chlorhexidine as a vehicle gives a synergistic antibacterial effect, although there are concerns about its ability to promote healing. CMCP and similar toxic phenolic agents are no longer recommended.

### Mechanism
Calcium hydroxide dissociates into calcium and hydroxyl ions, giving a pH of about 12.5. The high alkalinity gives a bactericidal effect, neutralises lipopolysaccharide, denatures necrotic tissue at the apex and creates a mildly necrotic zone in contact with the vital tissue; this irritation stimulates differentiation of cells in the periapical region into hard tissue-forming cells. The barrier produced is not true dentin, and is usually a mixture of cementum-like and osteodentin-like tissue, often with "Swiss cheese" porosities, so it is not leak-proof.

### Advantages
- Low cost, readily available and well known.
- Strong antimicrobial effect, useful in infected teeth.
- Long track record with predictable barrier formation, with reported success rates of the order of 74% to 100% in the literature, although mostly from case series.

### Disadvantages
- **Long treatment time and many visits**, which put a strain on compliance. Patients lost to follow-up are at risk of a leaky temporary seal and reinfection.
- **Weakening of root dentin.** Prolonged exposure to calcium hydroxide alters the mechanical properties of dentin, probably by hydrolysis and denaturation of the collagen matrix and the non-collagenous proteins. Andreasen and colleagues (2002) showed that the risk of cervical root fracture was higher in teeth with less root development and with longer periods of dressing. In an animal model, 12 months of calcium hydroxide dressing decreased fracture strength markedly, and the figure of about 50% is often quoted. Cvek (1992) also identified fracture as an important late complication after long-term treatment. This is probably the strongest argument against long-term calcium hydroxide apexification in very immature teeth.
- **Incomplete or porous barrier** with variable quality.
- **Coronal leakage** between visits, and reinfection.
- **Does not promote continued root lengthening or wall thickening.**
- Tunnel defects in the barrier.

### Current role
Calcium hydroxide remains useful as an interappointment disinfectant in the first one or two weeks, as a medicament in REP, and in settings where MTA or bioceramics are not available.

## 7.4 MTA apical barrier (apical plug)

### Background
Mineral trioxide aggregate was developed at Loma Linda University by Torabinejad in the 1990s as a root-end filling and perforation repair material. Its use as an apical plug in open apex teeth was reported by Shabahang and Torabinejad and others around 1999 to 2000. The plug provides an immediate artificial barrier against which gutta-percha or a bonded filling can be placed in one or two visits, avoiding the long delay and the weakening effect of calcium hydroxide.

### Technique (orthograde apical plug)
1. Access, working length, irrigation and (where infection is present) a short interval of calcium hydroxide, as above. After 1 to 2 weeks, check for resolution of symptoms. In vital or non-infected teeth a single visit is possible.
2. At the placement visit, remove the calcium hydroxide thoroughly by irrigation, ideally with passive ultrasonic activation, and 17% EDTA to remove residual paste from the walls (residual calcium hydroxide may interfere with sealing).
3. Dry the canal with paper points. If the canal bleeds persistently, a second brief application of calcium hydroxide may be needed.
4. **Matrix (optional but recommended in wide or resorbed apices).** An internal matrix prevents extrusion of the material into the periapical tissues. Choices include resorbable collagen (CollaPlug, CollaCote, Collatape), calcium sulfate, hydroxyapatite granules, or absorbable gelatin sponge. Place the matrix through the apex a millimetre or so and condense gently. Matrices can create a base for the plug, but none of them is mandatory in a case where the periapical tissues are already dry and the apical anatomy offers an acceptable stop. An alternative is to condense MTA carefully with no matrix when the apex is small.
5. **Mix the material** according to the manufacturer: ProRoot MTA powder to sterile water in a ratio of about 3:1 by volume, mixed to a thick, putty-like consistency.
6. **Deliver** the MTA into the canal using an MTA carrier (for example, the MAP system), a Lentulo, or a small amalgam carrier, and condense with pre-fitted pluggers or large paper points to produce a plug of 3 to 5 mm (4 to 5 mm in many protocols). Indirect ultrasonic vibration of the pluggers can improve adaptation. A radiograph confirms length and density.
7. **Moist cotton pellet** placed over the plug and the access sealed temporarily for 24 hours (or at least until setting; the original setting time of about 2 hours 45 minutes), because MTA needs moisture to set. A second visit is used to confirm that the plug is hard.
8. **Obturation of the remaining canal** with warm gutta-percha, thermoplasticised gutta-percha or a bonded resin, and sealer placed against the plug, followed by a bonded restoration. Alternatively, the cervical part of the root can be reinforced with a bonded composite, with or without a fibre post.
9. **Follow-up** at 3, 6 and 12 months, then yearly. The sign of healing is resolution of the periapical radiolucency and absence of symptoms.

### Outcomes
Case series and trials report high success in MTA apexification, with figures around 90% or above. Witherspoon and co-workers (2008) analysed 144 open-apex teeth obturated with an MTA plug, and reported about 93% healing. Systematic reviews generally conclude that MTA gives results comparable with calcium hydroxide for periapical healing, with a much shorter time to completion, although the evidence is of low to moderate certainty. A 2026 systematic review and meta-analysis of white MTA in REP and apexification is in the literature (PMID 42160814).

### Advantages
- Single or two-visit treatment.
- Predictable apical seal, with biocompatibility, hydrophilicity and ability to set in the presence of moisture and blood.
- Bioactivity: releases calcium ions and forms hydroxyapatite on contact with tissue fluids, and induces cementum-like deposition on its surface.
- Antibacterial effect from high pH and low solubility.
- Avoids prolonged weakening of dentin by calcium hydroxide.

### Disadvantages
- Does not allow continued root development.
- **Material handling** is difficult: it is granular, sandy, and not easily placed in a narrow canal.
- **Tooth discolouration** with grey MTA and with white MTA, which has been attributed to the bismuth oxide radiopacifier and contact with blood and with sodium hypochlorite or chlorhexidine; barrier should be placed no higher than the level of the apex region to limit crown contact.
- Long setting time and need for a second visit to confirm the set before continuing.
- Cost.
- Residual thin walls remain susceptible to fracture unless reinforced with bonded restoration.

## 7.5 Other apical barrier materials

### Biodentine
A tricalcium silicate-based dentin substitute. The powder contains tricalcium silicate, dicalcium silicate, calcium carbonate and oxide filler, and zirconium oxide as the radiopacifier. The liquid has calcium chloride (an accelerator) and a hydrosoluble polymer (a water-reducing agent). Setting time is about 9 to 12 minutes (initial), considerably quicker than MTA. It has good handling properties, higher compressive strength than MTA after setting, and does not discolour as much as MTA. It is used for pulp capping, pulpotomy, perforation repair, root-end filling and as an apical plug. Biodentine in the apical region is less well researched than MTA, and the plug should be adequately supported.

### Premixed bioceramics (EndoSequence BC RRM Putty and Fast Set Putty; TotalFill BC RRM)
Calcium silicate-based premixed putty with zirconium oxide, tantalum oxide, calcium phosphate monobasic. No mixing is needed, the material is ready to use from a syringe or jar, and it sets by hydration with moisture from the dentin. The fast-set version sets in about 20 minutes. Tantalum oxide shows less discolouration than bismuth oxide. These materials are easy to pack, and good clinical results have been reported, though the long-term data in apexification are still limited.

### Other MTA-type cements
- **MTA Angelus** (white and grey; calcium tungstate in white to avoid bismuth discolouration).
- **NeoMTA / NeoPutty** (tantalum oxide).
- **MTA Plus**, with a finer particle size and a liquid gel to improve handling.
- **Retro MTA** and **Bio-C Repair**.
- **Calcium-enriched mixture (CEM) cement**, developed in Iran by Asgary, composed of calcium oxide, sulfate, phosphate and silicate. It is sold as a root-end filling and apexification material, with reported success comparable to MTA in small trials.
- **Endocem**: a fast-setting, MTA-like Portland-cement material.
- **TheraCal LC**: a light-cured, resin-modified calcium silicate used as a pulp-capping liner (not an apical plug).

### Other historic materials
Tricalcium phosphate, freeze-dried bone and dentin chips, hydroxyapatite, osteogenic protein 1 and collagen-calcium phosphate gels have been tested as apical barriers or matrices, mostly in the 1970s to 1990s. They are of historical interest. Resorbable hydroxyapatite and calcium sulfate survive as internal matrices.

## 7.6 Obturation and restoration after barrier formation

### Obturation
Once the apical barrier is in place:
- Use warm vertical compaction or thermoplasticised injectable gutta-percha with a resin sealer. Take care to compact gently, since thin walls can be split by heavy lateral or vertical forces.
- Alternatively, in thin canals, use a custom-fitted master cone, or fill the root with a dentin-bonded composite for a "monoblock" effect.

### Root reinforcement
Because thin roots fracture, reinforcement is often recommended. Options include:
- Bonded composite resin placed inside the cervical and middle third of the root, using a bonding agent and light or dual cure composite, sometimes with glass fibre posts.
- Glass ionomer or resin-modified GIC as a base.
- Fibre-reinforced composite posts (glass or quartz), which have a modulus of elasticity closer to dentin than metal posts.
- Avoid rigid metal posts, which concentrate stress on a thin wall.

The essential principle is a coronal seal. Coronal leakage is a main cause of failure in endodontically treated teeth, and it is important to place a bonded restoration soon after obturation.

### Long-term restoration
The anterior tooth is restored with composite resin, and, if indicated, a crown after growth is complete. Because the gingival margin may shift during growth, definitive crowns are generally delayed until adulthood.

## 7.7 Surgical apexification and apical surgery

Surgical management is considered where non-surgical treatment has failed, the barrier cannot be placed from above (for example, due to a blocked canal, a separated instrument or a large lesion with persistent exudate), or an overextended material needs removal. It involves flap elevation, curettage of the lesion, root-end resection (minimal for short roots), and placement of a retrograde MTA or bioceramic filling. In an immature tooth with a short root, the resection further shortens the root, and the surgical approach should be a last resort.

# 8. Regenerative Endodontic Procedures

## 8.1 Historical background

The idea that an immature necrotic tooth could regain a blood supply goes back to Nygaard-Ostby (1961), who showed in experimental work that a blood clot induced in the canal of a pulpless tooth could be replaced by vascularised connective tissue. Iwaya and colleagues (2001) reported a case of an immature premolar with apical periodontitis and a sinus tract that was disinfected with a combination of ciprofloxacin and metronidazole and showed apical closure and root development. Banchs and Trope (2004) reported a similar case of an immature mandibular premolar with a sinus tract, using a triple antibiotic paste (ciprofloxacin, metronidazole and minocycline), induced bleeding and an MTA seal, and this paper is generally taken as the starting point of the modern protocol. Later case series (Jung et al., 2008) and the introduction of AAE (2007 onwards) and ESE (2016) guidelines established a standard approach. Hargreaves and colleagues (2013) set out the biological basis: disinfection, a scaffold, and signalling molecules with stem cells.

## 8.2 The tissue engineering triad

All REPs rely on three elements.

1. **Stem cells.** In REP these come from the periapical region. When bleeding is induced past the apex, mesenchymal stem cells from the apical papilla (SCAP), periodontal ligament stem cells, bone marrow stem cells and dental pulp remnants enter the canal in the blood clot. Lovelace and co-workers (2011) showed that evoked bleeding brings in a significant quantity of mesenchymal stem cell markers into the canal.
2. **Growth factors (signalling molecules).** Dentin is a reservoir of growth factors (TGF-beta1, BMPs, FGF-2, IGF, VEGF) that are released when the canal wall is conditioned with EDTA (Galler et al., 2011). Platelets in the clot or concentrate add PDGF, VEGF and TGF-beta.
3. **Scaffold.** The blood clot itself acts as the standard scaffold; alternatives are platelet-rich plasma (PRP), platelet-rich fibrin (PRF), platelet pellets, collagen, and various hydrogels.

A fourth requirement is **disinfection**, since the stem cells and growth factors are of no value in an infected canal, and a **coronal seal**, since leakage reinfects the canal.

## 8.3 Case selection

Case selection follows the AAE criteria already discussed: necrotic pulp, immature apex, no need for the canal space for a post, cooperative patient and parent, and no allergy to the medicaments used. Preferably the tooth has a root that is clearly shorter than its neighbour, with thin walls and a wide apical foramen (often cited as about 1 mm or more). It is the first choice in teeth with very immature roots (Cvek stage 1 to 3), where apexification would give a poor result.

Contraindications and cautions include: teeth with severe crown destruction that need a post for retention; teeth with complete or nearly complete roots (little to gain); vertical root fracture; severe external inflammatory or replacement root resorption; patients who cannot attend recall; patients with allergies to the antibiotic or the anaesthetic used; and anatomically complex teeth where disinfection cannot be achieved.

## 8.4 Step-by-step protocol (AAE 2021 and ESE 2016 consensus)

The protocol is that of the AAE Clinical Considerations for a Regenerative Procedure (revised 2021) and the ESE position statement (Galler et al., 2016), with differences noted.

### First appointment: disinfection

1. **Informed consent** as above, discussing alternatives.
2. **Local anaesthesia** with vasoconstrictor, **rubber dam isolation**, and **access** under magnification.
3. **Working length** estimated radiographically.
4. **Irrigation** with 20 mL of sodium hypochlorite per canal for about 5 minutes, using a closed-end needle with side vents or a negative pressure system (EndoVac) to reduce the risk of extruding irrigant into the periapical space. The AAE advises a lower concentration, 1.5% to 3%, to minimise cytotoxicity to stem cells in the apical tissues. The ESE recommends 1.5% NaOCl. The needle is placed approximately 1 mm from the root end (AAE) and, in other recommendations, 1 to 2 mm short of the working length.
5. **Rinse** with saline or EDTA (20 mL per canal, 5 minutes). Some operators use saline in the first appointment and keep EDTA for the second visit.
6. **Minimal or no mechanical instrumentation.** The walls are thin, and a primary goal is to preserve any remaining tissue and SCAP.
7. **Dry the canal** with paper points.
8. **Intracanal medicament:** either calcium hydroxide, or a low concentration triple antibiotic paste (TAP) or double antibiotic paste (DAP). Calcium hydroxide is the ESE choice and is the most widely used because it is easy, inexpensive, does not stain, and (if used without excess) is relatively kind to stem cells. The AAE document allows a low concentration of TAP; a consensus paper cites 1 to 5 mg/mL of antibiotic dressing for the AAE position (verify the exact figure in the current AAE document before quoting it). If TAP is used, the pulp chamber should be sealed, using a dentin bonding agent before placing the paste, to minimise discolouration. The medicament is placed below the CEJ, carried by a Lentulo or syringe, and not allowed to touch the coronal dentin.
9. **Temporary restoration** of 3 to 4 mm of a material such as Cavit, IRM or glass ionomer.
10. The patient is recalled in 2 to 4 weeks (some say 1 to 4 weeks, and ESE suggests up to 4 weeks).

### Second appointment: evoking bleeding and sealing

1. Assess the response to the first visit. If there are signs or symptoms of persisting infection (swelling, sinus tract, tenderness) the disinfection is repeated with a fresh medicament. Some clinicians use an alternative antibiotic paste at this step.
2. **Anaesthesia** with a local anaesthetic without a vasoconstrictor (3% mepivacaine plain is the AAE recommendation), because vasoconstrictors reduce the bleeding needed. Rubber dam and re-entry.
3. **Remove the medicament** by gentle irrigation with 17% EDTA (20 mL per canal for 5 minutes), with the needle positioned 1 to 2 mm from the root end, to remove the intracanal medicament and condition the dentin, releasing growth factors. Passive ultrasonic activation or other activation can be used to help removal of calcium hydroxide.
4. **Rinse with saline** (5 mL) to remove the EDTA, and dry with paper points.
5. **Induce bleeding.** A pre-curved sterile K-file (size 25 or larger) is rotated about 2 mm past the apical foramen to lacerate the periapical tissue and cause bleeding, filling the canal to a level 3 mm below the CEJ. Bleeding can be slow, and cotton pellets are used to adjust the level of the blood. If bleeding cannot be induced, the AAE says the procedure should be aborted or the patient be recalled and tried again, or the operator may switch to an apexification technique.
6. **Clot formation.** Wait for 15 minutes for a blood clot to form. A clot well formed at the level 3 mm below the CEJ provides a base for the coronal seal.
7. **Scaffold addition (optional).** A resorbable collagen matrix (CollaPlug, CollaCote, CollaTape) may be placed over the clot to support the material. PRP or PRF can be placed in place of, or in addition to, the blood clot.
8. **Coronal seal.** Place 3 to 4 mm of a calcium silicate barrier (white MTA, Biodentine or an equivalent bioceramic) directly on the collagen matrix or clot, with a moistened cotton pellet when MTA is used. A thick layer of glass ionomer (resin-modified) can be placed over it, and the tooth is restored with a bonded composite.
9. **Alternatives** include the placement of a bioceramic putty directly, and light-cured glass ionomer immediately over Biodentine to allow a single-visit restoration.

The location of the barrier is chosen at or just below the CEJ, which has two benefits: it reduces crown staining by the MTA, and it allows the maximum amount of canal space for tissue growth.

### Follow-up

The tooth is evaluated clinically and radiographically at 3, 6, 12 and 18 to 24 months, and then yearly. The AAE advises a minimum of 1 year (ideally 4 years or longer). Radiographs should be taken with a standardised angulation and, where practical, with a positioner so that root length and wall thickness changes can be measured.

## 8.5 Irrigants in REP

The irrigants must disinfect the canal in the absence of mechanical cleaning, without destroying the SCAP and the growth factors.

- **Sodium hypochlorite.** The best antimicrobial and tissue-dissolving irrigant. High concentrations (5.25% to 6%) reduce SCAP survival and differentiation. Martin and co-workers (2014) found that 6% NaOCl was strongly detrimental to the survival of SCAP, 3% was less so, and 1.5% was compatible when followed by 17% EDTA, which is the basis for the 1.5% to 3% recommendation. The ESE suggests 1.5% for 20 mL per canal. Using a closed-end side-vented needle at a safe distance and limiting the volume reduces the risk of apical extrusion, which can cause severe pain, swelling and damage to the stem cell population.
- **EDTA 17%.** A chelator which removes the smear layer, and demineralises dentin to release embedded growth factors, with an effect on cell attachment and differentiation. Dentin conditioning with 17% EDTA has been shown to increase the release of TGF-beta1 and to favour SCAP survival and attachment (Galler et al., 2011). Alternatives under investigation include 10% citric acid, which has been reported to release more TGF-beta1 in some in-vitro work, and etidronic acid (HEDP), which in a clinical trial gave more post-operative pain than EDTA or citric acid.
- **Chlorhexidine.** Not advised as the main irrigant for REP. It is toxic to stem cells, does not dissolve tissue, and reacts with hypochlorite. A low concentration may be considered as a final rinse by some authors, but it is not part of the AAE or ESE protocols.
- **Saline.** Used as an intermediate rinse to prevent mixing of hypochlorite and EDTA or chlorhexidine.
- **Activation.** Ultrasonic activation, sonic activation and negative pressure systems may improve the efficacy of disinfection, and laser or photodynamic therapy have been studied as adjuncts, but the evidence is still limited.

## 8.6 Intracanal medicaments

### Triple antibiotic paste (TAP)
Equal parts of ciprofloxacin, metronidazole and minocycline, originally developed by Hoshino and colleagues as "3Mix-MP" for lesion sterilisation and tissue repair (LSTR) in primary teeth. The mixture covers a broad spectrum including anaerobes and Gram-negative bacteria in dentinal tubules. High concentrations (about 1 mg/mL to 1000 mg/mL) have been shown in laboratory studies to be toxic to SCAP, so the dilute form is advised, at a concentration of 0.1 mg/mL to 1 mg/mL in several sources. The main problem is **crown discolouration** from minocycline, which binds calcium in dentin and forms a chelate which stains it grey or dark. Strategies are to replace minocycline with a different antibiotic (cefaclor, clindamycin, amoxicillin, or fosfomycin for the penicillin-allergic), to seal the dentin with a bonding agent before placing the paste, to keep the paste in the root and below the CEJ, and to use a dentin sealing agent.

### Double antibiotic paste (DAP)
Ciprofloxacin and metronidazole alone. It lowers the risk of discolouration and is considered at least as effective in clinical series. Use of DAP is permitted in the AAE documents.

### Calcium hydroxide
At a pH of about 12.5, it is an effective intracanal antimicrobial and also promotes the release of growth factors from dentin (a short exposure). It does not discolour the crown and is favoured by the ESE. Over a long period (months) or at high concentration it can reduce SCAP survival and harm the dentin structure, so a short exposure of 2 to 4 weeks is the rule. Residual calcium hydroxide must be fully removed before bleeding is induced, because it can interfere with clot formation and cell attachment.

### Other options studied
Antibiotic pastes in a slow-release polymer, chitosan or nanoparticle carriers, and ledermix-like pastes have been proposed. Evidence in humans is limited.

## 8.7 Scaffolds in REP

### Blood clot
The standard scaffold in AAE and ESE protocols. Its advantages are that it is autologous, simple, inexpensive, and rich in growth factors, stem cells and fibrin. Its disadvantages are that it is variable, difficult to obtain in some canals, and may not remain in place when the canal is dry.

### Platelet-rich plasma (PRP)
Autologous plasma with a high concentration of platelets, prepared by drawing venous blood, centrifugation and activation with calcium chloride. Torabinejad and Turman (2011) described its use in a necrotic open-apex tooth. PRP can be placed in the canal after a blood clot is formed, or as a replacement. It needs a blood draw and equipment, which is a nuisance in a young child.

### Platelet-rich fibrin (PRF)
A second-generation platelet concentrate made without anticoagulants, by centrifuging blood to give a fibrin matrix rich in platelets, leukocytes and growth factors that are released slowly. PRF is a more stable scaffold than PRP and does not need exogenous activating agents. Studies (including Keswani and Pandey, 2013) report successful use in necrotic immature teeth. Variants include i-PRF and A-PRF.

### Platelet pellet
Blood is spun to form a platelet pellet that is placed in the canal, and it showed good results in some trials.

### Collagen and gelatin
Resorbable collagen (CollaPlug) used over the clot as a matrix to hold the barrier material. Collagen has been combined with bFGF.

### Other scaffolds and hydrogels
Chitosan, hyaluronic acid, self-assembling peptide hydrogels (Puramatrix), gelatin methacrylate, alginate, and synthetic polymers such as polylactic acid have been tested in the laboratory and animals, with a few human reports. These offer a more controlled architecture and the capacity to release drugs or growth factors.

### What does the evidence say about scaffolds?
A systematic review and network meta-analysis (Sabeti et al., 2024; PMID 37966465) of 9 randomised controlled trials compared blood clot, blood clot with bFGF, blood clot with collagen, platelet pellet, PRP and PRF. PRP showed the greatest increase in root length at 6 to 12 months, and PRP or PRF showed the highest success at up to 12 months, but the certainty of evidence was very low to low. Beyond 12 months, blood clot with collagen or with bFGF ranked highest for the secondary outcomes. The authors concluded that PRP and PRF seem useful in the short term, not in the long term, and that more randomised trials are needed.

## 8.8 Coronal barrier materials in REP

The coronal seal is placed on the clot or collagen and must be biocompatible, bioactive and able to set in the presence of moisture.

- **White MTA** (ProRoot, MTA Angelus white) is the longest-established and has good evidence. Grey MTA is avoided in the aesthetic zone.
- **Biodentine** is a rapidly setting alternative with less discolouration risk. Some in-vitro work shows that it has a favourable effect on SCAP viability compared with MTA.
- **EndoSequence BC RRM putty** and other premixed bioceramics, with tantalum oxide, avoid discolouration from bismuth.
- **Glass ionomer** (resin-modified) is placed over the barrier as the base, and a composite restoration is bonded over it. The final restoration should be placed promptly to maintain the coronal seal.
- A **sealed restoration** is probably as important as the identity of the barrier. Coronal microleakage is a recognised cause of failure.

## 8.9 Outcomes of REP

### Outcome criteria (Wigler et al., 2013 and AAE)
- **Primary goal:** elimination of symptoms and evidence of bony healing. This is the minimum requirement for success.
- **Secondary goal (desirable):** increased root wall thickness and/or increased root length.
- **Tertiary goal:** a positive response to pulp sensibility testing, indicating a functional pulp.

### Radiographic patterns
Chen and co-workers (2012) classified radiographic outcomes: continued thickening of the walls and root maturation; no significant root development with blunting or closure of the apex; continued apical closure without wall thickening; severe canal calcification; and a hard tissue barrier forming between the coronal MTA and the apex. Responses vary among teeth, even in the same patient and protocol.

### What tissue forms? Histology
Animal and human histological studies (including experiments in dogs and ferrets, and human teeth extracted later for other reasons) show that the new tissue in the canal is generally **not** pulp. Instead, cementum-like tissue, bone-like tissue and periodontal ligament-like connective tissue grow into the canal and deposit along the walls. The process is therefore best called **repair** or "regeneration of non-pulp tissue", although it often gives clinical benefits (continued root development, healing, sometimes a positive sensibility test). Odontoblast-like cells and true pulp-dentin regeneration have been reported only occasionally.

### Clinical results
Reviews report high survival and healing rates (generally 90% or above) for REP in necrotic immature teeth. In the 2025 umbrella review (Tewari et al., PMID 39692071) of 12 systematic reviews, the success and survival rates of REP and apexification were comparable (more than 85% in all domains), and REP was superior for increasing root thickness and length, but the evidence was weak and most of the reviews were of low or critically low quality. In practical terms, REP offers a biological advantage in root development but has no proven advantage in tooth survival over a well-performed MTA apexification.

### Failures and complications
- Persistent or recurrent infection (poor disinfection or leaking coronal seal).
- No bleeding can be induced.
- Crown discolouration (minocycline, MTA).
- Intracanal calcification and canal obliteration, which makes retreatment very difficult.
- Lack of root development or only apical closure.
- Pain and swelling, which may result from irrigant extrusion.
- Root resorption, replacement resorption or ankylosis.
- Allergic reaction to an antibiotic.
- Cervical root fracture: lower with REP than with long-term calcium hydroxide, but still possible if walls remain thin.

### What to do if REP fails
The options are repeat disinfection and a second REP, conversion to MTA/bioceramic apexification, surgical endodontics, or extraction. Because a failed REP may leave a calcified canal, apexification should be carried out promptly while the canal can still be negotiated.

## 8.10 Single-visit and modified protocols

Some case reports describe single-visit REP using only copious irrigation with NaOCl and EDTA, activation and no medicament. Advocates cite fewer visits and no staining. The evidence base is thin, and most guidelines prefer two visits and an interappointment medicament. Other modifications include using laser disinfection, adding PRF, mixing bioceramics into the scaffold, or treating immature molars with similar protocols.

# 9. Materials Compared

## 9.1 Summary table

| Material | Composition and key features | Main uses in immature teeth | Strengths | Limitations |
| Calcium hydroxide | Ca(OH)2, pH about 12.5, in water, saline or oil/iodoform vehicle | Long-term apexification, interappointment dressing, REP medicament | Cheap, antibacterial, well documented | Long treatment, weakens dentin, porous barrier |
| MTA (grey, white) | Portland cement, tricalcium and dicalcium silicate, bismuth oxide | Apical plug, pulp capping, pulpotomy, REP coronal seal | Biocompatible, sealing, bioactive, strong evidence | Staining, slow set, difficult handling, cost |
| Biodentine | Tricalcium silicate, zirconium oxide, calcium chloride liquid | Pulpotomy, pulp capping, apical plug, REP barrier | Set in 9 to 12 min, good handling, less staining | Less long-term apical data |
| EndoSequence BC RRM / TotalFill | Calcium silicates, zirconium and tantalum oxide, premixed | Apical plug, perforation, REP barrier | Premixed, easy, less staining | Limited long-term data, cost |
| CEM cement | Calcium oxide, sulfate, phosphate, silicate | Pulp capping, apical plug | Fast set, good seal | Fewer clinical studies |
| Triple antibiotic paste | Ciprofloxacin, metronidazole, minocycline | REP medicament | Broad spectrum, dentinal tubules | Staining, cytotoxic at high dose, allergy |
| Double antibiotic paste | Ciprofloxacin, metronidazole | REP medicament | Less staining | Narrower spectrum |
| Collagen matrix (CollaPlug etc.) | Resorbable bovine collagen | Matrix for plug, REP support | Prevents extrusion, holds barrier | Does not itself regenerate tissue |
| PRP, PRF | Autologous platelet concentrates | REP scaffold | Growth factors, autologous | Needs blood draw, variable protocols |
| Resin-modified GIC | Glass ionomer with resin | Base over barrier, reinforcement | Chemical bond to dentin, fluoride | Not a barrier by itself |
| Bonded composite and fibre posts | Resin composite, glass fibre | Root reinforcement, final restoration | Strengthens thin walls | Technique-sensitive, needs isolation |

## 9.2 Properties of an ideal apical barrier material

An ideal material should be biocompatible, bioactive (promoting hard tissue deposition), antibacterial, dimensionally stable, resistant to washout, radiopaque, easy to place, able to set in the presence of moisture and blood, non-staining and inexpensive. No available material meets all these criteria. Calcium silicate cements meet most of them, which is why they have replaced calcium hydroxide as the standard for apical plugs and coronal seals.

## 9.3 MTA versus Biodentine versus premixed bioceramics

- **Setting time:** MTA about 2 hours 45 minutes (for ProRoot), Biodentine around 12 minutes, BC putty about 30 minutes (fast set about 20 minutes).
- **Handling:** MTA is sandy and needs a carrier. Biodentine is more cohesive, and putty types are ready to use.
- **Colour stability:** Bismuth oxide leads to grey staining, especially in contact with NaOCl and blood. Zirconium oxide and tantalum oxide are more stable.
- **Strength:** Biodentine reaches higher compressive strength sooner. All are low in tensile strength compared to resin composites, so they are not a structural filler.
- **Evidence:** MTA has far more clinical data, and many newer materials rely on in-vitro and short-term clinical studies.
- **Biological effect on SCAP:** In-vitro studies show good viability and differentiation with MTA, Biodentine and bioceramic putty, each releasing calcium ions and favouring odontoblastic differentiation.

Successfully replaced 1 instance(s) of the string in '/tmp/workspace/immature-apex/src/part3.txt'

# 10. Emerging and Experimental Approaches

## 10.1 Cell-based therapy

The standard REP is a "cell homing" approach that relies on the patient's own periapical cells. Cell-based approaches deliver stem cells into the canal on a scaffold. Candidate cells include dental pulp stem cells (DPSC), stem cells from human exfoliated deciduous teeth (SHED), SCAP, periodontal ligament stem cells and bone marrow or adipose-derived stem cells. Xuan and colleagues (2018) reported a small clinical trial in which autologous stem cells from deciduous teeth were implanted into children's injured immature permanent incisors, with evidence of pulp-like tissue formation and increased root length and apical closure compared with apexification controls. The study was small and early, and the approach faces regulatory, cost and logistic barriers: cell harvesting, culture, good manufacturing practice facilities and long-term safety.

## 10.2 Cell homing and bioactive molecules

Instead of delivering cells, the scaffold is loaded with chemotactic factors (for example SDF-1, bFGF, VEGF, BMP-7, NGF) that recruit host stem cells to the canal. Controlled release is achieved with microspheres or hydrogels. Combined scaffolds with collagen and bFGF have shown effects in clinical trials, as the network meta-analysis above notes. Other products under investigation include exosomes derived from stem cells, simvastatin or other small molecules incorporated in scaffolds, and mineral trioxide aggregate or bioceramic particles that release ions to stimulate odontoblast differentiation.

## 10.3 Injectable and printed scaffolds

Injectable hydrogels (self-assembling peptides, hyaluronic acid, fibrin, chitosan and gelatin methacrylate) can fill the canal with complex geometry and carry cells and drugs. Three-dimensional bioprinting and electrospun nanofibre scaffolds are being developed to make scaffolds that mimic the architecture of pulp and dentin. All remain at the laboratory or animal stage.

## 10.4 Disinfection innovations

Studies are looking at antibiotic-loaded nanofibres and polymer carriers, antibiotic pastes with better biocompatibility, photodynamic therapy, diode and Nd:YAG lasers, ozone, and chitosan nanoparticles, with the aim of disinfecting thoroughly without harming stem cells. Their results are still preliminary.

## 10.5 Gene therapy and others

Gene delivery of growth factors, and tooth bioengineering from germ-like cell aggregates, are research-stage possibilities. None is available for clinical use.

# 11. Special Clinical Situations

## 11.1 Traumatised teeth in the IADT guidelines

The International Association of Dental Traumatology (IADT) guidelines (2020) give guidance on pulp management after luxation, fracture and avulsion.

- **Complicated crown fracture, immature tooth:** pulp capping or partial pulpotomy is the treatment of choice to preserve vitality and allow continued root development; pulpectomy or apexification/REP if pulp is necrotic.
- **Luxation injuries:** immature teeth have a good chance of pulp revascularisation, particularly after extrusive and lateral luxation with open apices. Pulp extirpation is not done immediately. The tooth is monitored clinically and radiographically; if necrosis and infection develop (discolouration alone is not sufficient; signs include a sinus tract, periapical radiolucency or inflammatory resorption), REP or apexification is performed.
- **Intrusion:** very high risk of necrosis and inflammatory resorption; endodontic intervention is usually required within 2 to 3 weeks in mature teeth, but in immature teeth spontaneous eruption and revascularisation are monitored.
- **Avulsion, immature tooth:** immediate replantation gives the best chance of revascularisation. Topical doxycycline or minocycline before replantation, and systemic antibiotics, are recommended by IADT for open-apex teeth. If the extra-alveolar time is long (dry time over 60 minutes), the periodontal ligament is nonviable; if the tooth was replanted, root canal treatment is planned when infection or resorption appears, and in children the tooth may be treated mainly to maintain alveolar bone and delay ankylosis consequences until growth is complete.
- **Root fracture:** a root fracture in an immature tooth often heals with a hard tissue callus. Pulp necrosis occurs in the coronal fragment only in a minority and is treated by apexification or REP in the coronal segment.

## 11.2 Teeth with large apical lesions or sinus tracts

Both REP and apexification are possible with large lesions. Disinfection is the key, and the interappointment medicament should be changed or extended if the sinus tract persists. An overlong period of calcium hydroxide is avoided in very immature roots because of the fracture risk.

## 11.3 Dens evaginatus and dens invaginatus

- **Dens evaginatus:** necrosis with an open apex is typical. Prophylactic reinforcement of the tubercle with composite before root completion is recommended. Both REP and apexification have been reported, with REP preferred in very immature roots. Pulp therapy (partial pulpotomy) can be tried if the pulp is vital after tubercle fracture.
- **Dens invaginatus (Oehlers type II and III):** anatomy is complex and disinfection is the challenge. Passive ultrasonic activation, CBCT and microscopy are valuable. REP in these teeth has been reported but depends on the ability to disinfect the invagination.

## 11.4 Immature molars and premolars

Evidence from case series suggests that REP can be used in immature molars and premolars, with higher rates of persistent infection in multi-canal teeth because of anatomical complexity. Pulpotomy (partial or full) with MTA or Biodentine is more predictable for vital immature molars. If the pulp is necrotic, either technique can be used, but the pulp chamber floor should not be thinned to place a post, because a barrier is placed at the canal orifices.

## 11.5 Resorption

Inflammatory external resorption in an immature tooth with necrosis is arrested by thorough disinfection and calcium hydroxide, then MTA/bioceramic apexification or REP. Replacement resorption (ankylosis) cannot be treated endodontically; the problem is infraposition in a growing child, which is managed by monitoring, decoronation or extraction at the right time. Internal resorption is treated by complete disinfection and, where possible, obturation, but it is a contraindication to VPT.

## 11.6 Medically compromised and allergic patients

Antibiotic paste may be contraindicated in patients with allergy to the component drugs (penicillin and cephalosporin allergy matters if substitutions are used). Calcium hydroxide alone is then used. Patients at risk of infective endocarditis require the usual consideration for antibiotic prophylaxis, though the evidence for it in endodontics is limited. Patients with poor compliance are better treated by a short, single-barrier apexification.

# 12. Comparison of the Three Approaches

| Feature | Vital pulp therapy (apexogenesis) | Apexification (Ca(OH)2 or MTA) | Regenerative endodontics |
| Pulp status | Vital | Necrotic | Necrotic |
| Aim | Preserve pulp, complete root | Induce or create apical barrier | Re-establish vascularised tissue, continued root growth |
| Root development | Yes | No | Often (variable) |
| Visits | 1 | Ca(OH)2: many; MTA: 2 to 3 | 2 plus recalls |
| Mechanical instrumentation | None to minimal | Minimal | None to minimal |
| Main materials | MTA, Biodentine, bioceramics | Ca(OH)2, MTA, Biodentine, bioceramic putty | NaOCl, EDTA, Ca(OH)2 or antibiotic paste, blood clot, PRF/PRP, MTA |
| Fracture risk | Lowest | Highest with long Ca(OH)2 | Intermediate |
| Staining risk | Low to moderate | Moderate with MTA | Moderate to high (TAP, MTA) |
| Evidence | Good for traumatic exposure | Moderate | Moderate, growing |
| When chosen | Pulp alive | Post needed, poor compliance, REP failed | Very immature root with infection, favourable case |

# 13. Evidence Review

## 13.1 Regenerative versus apexification

The most recent high-level evidence comes from systematic reviews. An umbrella review in Dental Traumatology (Tewari et al., 2025; PMID 39692071) of 12 systematic reviews found comparable clinical, radiographic and overall success (above 85%) for REP and apexification. REP gave superior root thickening and lengthening, while weak evidence favoured the apical plug technique for clinical and radiographic success. Only one review was of high quality, and the strength of evidence was graded weak. A network meta-analysis in International Endodontic Journal (Sabeti et al., 2024; PMID 37966465) compared scaffolds, with PRP and PRF doing best at up to 12 months, and blood clot with collagen or bFGF doing best beyond 12 months, all with very low to low certainty. A 2026 systematic review and meta-analysis in International Dental Journal (PMID 42160814) addressed white MTA in REP and apexification. I have not read the full text of the 2026 paper and cannot summarise its findings here.

## 13.2 Interpreting the evidence

Several caveats apply when presenting this evidence.

- **Heterogeneity.** Protocols differ in irrigants, medicaments, scaffolds and barriers, and definitions of success differ (clinical healing only, or also root development).
- **Short follow-up.** Many studies stop at 12 to 24 months, whereas the survival of a tooth is measured over decades.
- **Selection.** REP is applied to teeth with the most immature roots, whereas apexification is often used for teeth with more developed roots, which leads to bias in comparisons.
- **Surrogate outcomes.** Root lengthening and wall thickening are surrogate outcomes; whether they make the tooth more fracture resistant over the long term has not been proven in humans.
- **Quality.** Most trials have a risk of bias, and most reviews are of low quality.

A balanced conclusion for a seminar: REP is justified in suitably selected, very immature necrotic teeth because it has a plausible biological advantage and similar survival; apexification with a bioceramic plug remains a reliable and predictable alternative, particularly when a post is needed, compliance is limited, or REP has failed.

## 13.3 Calcium hydroxide versus MTA

Reviews consistently show that MTA gives similar or slightly better rates of periapical healing with fewer visits and shorter time to completion, and avoids long-term dentin weakening. That is why long-term calcium hydroxide apexification is now seldom the first choice. Differences in the rate of barrier formation and success between MTA and Biodentine are small in the few available trials.

## 13.4 Vital pulp therapy evidence

For traumatic exposures in immature incisors, Cvek's partial pulpotomy has a success rate of the order of 90% to 96% over years, and subsequent studies using MTA or Biodentine report similar results. For carious exposures in immature molars, meta-analyses show high success for pulpotomy with calcium silicates, and growing acceptance of the technique in teeth with symptoms of irreversible pulpitis.

# 14. Complications and Their Management

| Complication | Cause | Prevention and management |
| Persistent infection | Incomplete disinfection, coronal leakage | Adequate irrigation, medicament, good seal; repeat disinfection |
| Irrigant extrusion | Close needle placement, high pressure | Closed-end side-vented needle, short of the apex, slow flow |
| Crown discolouration | Minocycline, bismuth oxide, blood in dentin | Use DAP or Ca(OH)2, seal dentin, place MTA below CEJ, choose non-bismuth material |
| Cervical root fracture | Thin walls, long Ca(OH)2, trauma | Avoid prolonged Ca(OH)2, reinforce with bonded composite, avoid rigid posts |
| MTA/material extrusion | Wide apex | Internal matrix, careful condensation |
| Failure to evoke bleeding | Necrotic tissue, anaesthetic with vasoconstrictor | Plain anaesthetic, repeat visit, convert to apexification |
| Canal calcification | Over-stimulated deposition | Radiographic monitoring; difficult retreatment |
| Allergic reaction | Antibiotic paste | Take history; use Ca(OH)2 |
| Resorption | Infection, injury | Disinfection, early intervention |
| Lack of root development | Scar tissue, residual infection | Accept if healed; otherwise retreat |

# 15. Restoration and Follow-Up

## 15.1 Final restoration

A well-bonded coronal restoration is as important as the endodontic technique. After REP the coronal MTA/bioceramic is covered with glass ionomer, then composite. After apexification, a bonded composite or resin core, with or without a fibre post, reinforces the cervical root. In anterior teeth, aesthetic issues such as crown discolouration may be treated by internal or external bleaching, but with care: internal bleaching of a tooth with a thin cervical wall risks cervical resorption and fracture and should be done after root maturity, using the walking bleach method with a sealing base and avoiding heat.

## 15.2 Follow-up schedule

- VPT: 1, 3, 6, 12 months, then yearly for at least 4 years.
- Apexification: 3, 6, 12 months, then yearly.
- REP: 3, 6, 12 and 18 to 24 months, then yearly, with the AAE and ESE advising radiographic review for at least 1 year, and ideally up to 4 years or longer.

At every visit: pain, swelling, sinus tract, percussion, mobility, colour, sensibility response, and a radiograph to check healing of the periapical lesion, root length, wall thickness, and apical closure. A tooth that is clinically healthy and radiographically improving needs no additional treatment, whatever the extent of root development.

## 15.3 Long-term considerations

Children grow. Teeth treated before the end of growth may be affected by vertical facial growth and infraposition (for ankylosed teeth). Definitive prosthetic treatment, such as veneers and crowns, is postponed until growth is complete. Parents need to be told that the tooth is in a "treated but monitored" state for years.

# 16. Conclusion

An immature root apex changes the aims of endodontic treatment. The first priority is to preserve the vital pulp where possible, through pulp capping, partial pulpotomy or pulpotomy, because that is the only approach that reliably completes root formation. When the pulp is necrotic, the clinician chooses between apexification with a bioceramic plug and a regenerative endodontic procedure. Long-term calcium hydroxide apexification has largely been replaced because of its length and its weakening effect on dentin, but calcium hydroxide remains a useful medicament. MTA and newer calcium silicate materials (Biodentine, premixed bioceramics) have become the standard for barriers and coronal seals. REP is a conservative, biologically oriented option that depends on careful, gentle disinfection (1.5% to 3% NaOCl, 17% EDTA, calcium hydroxide or dilute antibiotic paste), an induced blood clot or platelet concentrate scaffold, and a good coronal seal. It can promote root lengthening and wall thickening, but the tissue is mostly repair tissue, the outcomes are variable, and the evidence is of low to moderate quality. Current evidence shows similar survival rates for the two approaches, so the choice should be individualised, using case selection, patient factors and operator skill.

# 17. Key Points for Revision and Viva

1. Define apexogenesis, apexification and REP, and state that apexogenesis needs vital radicular pulp.
2. Cvek stages 1 to 5 of root development, and why fracture risk is greatest in stage 1.
3. Why do thin walls and a wide apex complicate conventional obturation?
4. SCAP, HERS and the apical papilla blood supply: the biological basis of REP.
5. Pulp sensibility tests are unreliable in immature teeth; compare with the contralateral tooth.
6. Cvek pulpotomy: removal of 2 to 3 mm, haemostasis with NaOCl, MTA or Biodentine cap, bonded restoration.
7. Long-term Ca(OH)2: pH 12.5, 5 to 20+ months, weakens dentin, fracture risk (Andreasen et al., 2002).
8. MTA plug: 3 to 5 mm, moist cotton pellet, setting time about 2 hours 45 minutes, discolouration from bismuth.
9. Biodentine: tricalcium silicate, zirconium oxide, sets in about 12 minutes.
10. AAE REP protocol: visit 1 (1.5% to 3% NaOCl 20 mL, saline or EDTA, medicament); visit 2 (plain 3% mepivacaine, 17% EDTA, induced bleeding to 3 mm below CEJ, collagen optional, 3 to 4 mm MTA, bonded restoration).
11. Why minocycline stains and how to avoid it.
12. Outcome goals of REP: primary (symptoms and healing), secondary (root growth), tertiary (vitality response).
13. REP produces repair tissue (cementum-like, bone-like, PDL-like), not true pulp.
14. Evidence: success above 85% for both, REP better for root lengthening and thickening, weak evidence overall.
15. Avulsed immature tooth: immediate replantation, revascularisation possible.

# 18. Selected References

Reference details were assembled from memory and from search results, and should be checked against the original articles before they are used in a formal presentation.

1. American Association of Endodontists. AAE Clinical Considerations for a Regenerative Procedure. Revised 2021. Chicago: AAE.
2. Galler KM, Krastl G, Simon S, et al. European Society of Endodontology position statement: revitalization procedures. Int Endod J. 2016;49:717-723.
3. Tewari N, Devi P, Sampath S, et al. Comparative effectiveness of regenerative endodontic treatment versus apexification for necrotic immature permanent teeth with or without apical periodontitis: an umbrella review. Dent Traumatol. 2025. PMID 39692071.
4. Sabeti M, Ghobrial D, Zanjir M, et al. Treatment outcomes of regenerative endodontic therapy in immature permanent teeth with pulpal necrosis: a systematic review and network meta-analysis. Int Endod J. 2024. PMID 37966465.
5. Li Z, Xu Y, An S. Efficacy of white mineral trioxide aggregate in regenerative endodontic procedures and apexification for immature permanent teeth: a systematic review and meta-analysis. Int Dent J. 2026. PMID 42160814.
6. Banchs F, Trope M. Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod. 2004;30:196-200.
7. Hargreaves KM, Diogenes A, Teixeira FB. Treatment options: biological basis of regenerative endodontic procedures. J Endod. 2013;39(3 Suppl):S30-S43.
8. Galler KM, D'Souza RN, Federlin M, et al. Dentin conditioning codetermines cell fate in regenerative endodontics. J Endod. 2011;37:1536-1541.
9. Martin DE, De Almeida JF, Henry MA, et al. Concentration-dependent effect of sodium hypochlorite on stem cells of apical papilla survival and differentiation. J Endod. 2014;40:51-55.
10. Sonoyama W, Liu Y, Yamaza T, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth. J Endod. 2008;34:166-171.
11. Wigler R, Kaufman AY, Lin S, et al. Revascularization: a treatment for permanent teeth with necrotic pulp and incomplete root development. J Endod. 2013;39:319-326.
12. Chen MY, Chen KL, Chen CA, et al. Responses of immature permanent teeth with infected necrotic pulp tissue and apical periodontitis/abscess to revascularization procedures. Int Endod J. 2012;45:294-305.
13. Torabinejad M, Turman M. Revitalization of tooth with necrotic pulp and open apex by using platelet-rich plasma: a case report. J Endod. 2011;37:265-268.
14. Xuan K, Li B, Guo H, et al. Deciduous autologous tooth stem cells regenerate dental pulp after implantation into injured teeth. Sci Transl Med. 2018;10:eaaf3227.
15. Andreasen JO, Farik B, Munksgaard EC. Long-term calcium hydroxide as a root canal dressing may increase risk of root fracture. Dent Traumatol. 2002;18:134-137.
16. Cvek M. A clinical report on partial pulpotomy and capping with calcium hydroxide in permanent incisors with complicated crown fracture. J Endod. 1978;4:232-237.
17. Cvek M. Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha. Endod Dent Traumatol. 1992;8:45-55.
18. Frank AL. Therapy for the divergent pulpless tooth by continued apical formation. J Am Dent Assoc. 1966;72:87-93.
19. Rafter M. Apexification: a review. Dent Traumatol. 2005;21:1-8.
20. Witherspoon DE, Small JC, Regan JD, Nunn M. Retrospective analysis of open apex teeth obturated with mineral trioxide aggregate. J Endod. 2008;34:1171-1176.
21. Parirokh M, Torabinejad M. Mineral trioxide aggregate: a comprehensive literature review. Part I: chemical, physical, and antibacterial properties. J Endod. 2010;36:16-27.
22. Bourguignon C, Cohenca N, Lauridsen E, et al. International Association of Dental Traumatology guidelines for the evaluation and management of traumatic dental injuries. 1. Fractures and luxations. Dent Traumatol. 2020;36:314-330.
23. Fouad AF, Abbott PV, Tsilingaridis G, et al. International Association of Dental Traumatology guidelines for the evaluation and management of traumatic dental injuries. 2. Avulsion of permanent teeth. Dent Traumatol. 2020;36:331-342.
24. Kim SG, Malek M, Sigurdsson A, Lin LM, Kahler B. Regenerative endodontics: a review of current status and a call for action. Int Endod J. 2018;51:1367-1388.