What the future holds for regenerative endodontics: novel antimicrobials and regenerative strategies.

Regenerative/revitalisation endodontic techniques are increasingly used as a treatment approach for the management of immature permanent teeth with necrotic pulps. Different chemical irrigants and medicaments are routinely used clinically for intra-canal disinfection. However, despite remarkable progress in this field, coronal discolouration, cell cytotoxicity, difficulty of removal of organic biofilm from the root canal, development of sensitisation and antimicrobial resistance are still challenges to this line of treatment. This review critically discusses and challenges the current status quo of antimicrobials used in regenerative endodontics and sheds the light on future alternative antimicrobial materials with regenerative potential.

Investigating the vascularization capacity of a decellularized dental pulp matrix seeded with human dental pulp stem cells: in vitro and preliminary in vivo evaluations.

AIM: To investigate the vascularization capacity of a decellularized dental pulp matrix (DDP) of bovine origin seeded with human dental pulp stem cells (hDPSCs) in vitro and to present preliminary in vivo findings. METHODOLOGY: Bovine dental pulps were decellularized and then analysed using histological staining and DNA quantification. The resultant DDPs were characterized using immunohistochemical staining for the retention of vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF-2). Furthermore, DDPs were recellularized with hDPSCs and analysed histologically. The expression of markers involved in angiogenesis by hDPSCs colonizing the DDPs was assessed in vitro. A preliminary in vivo study was then conducted in which hDPSCs-seeded and unseeded DDPs were inserted in debrided human premolars root slices and implanted subcutaneously in immunodeficient mice. Samples were retrieved after 30 days and analysed using histological and immunohistochemical staining. The independent samples t-test, analysis of variance and a Kruskal-Wallis test were used to analyse the quantitative data statistically depending on the group numbers and normality of data distribution. The difference between the groups was considered significant when the P-value was less than 0.05. RESULTS: Acellular dental pulp matrices were generated following bovine dental pulp decellularization. Evaluation of the developed DDPs revealed a significant DNA reduction (P < 0.0001) with preservation of the native histoarchitecture and vasculature and retention of VEGF-A and FGF-2. Upon recellularization of the DDPs with hDPSCs, the in vitro analyses revealed cell engraftment with progressive repopulation of DDPs' matrices and vasculature and with enhanced expression of markers involved in angiogenesis. In vivo implantation of root slices with hDPSCs-seeded DDPs revealed apparent vascularization enhancement as compared to the unseeded DDP group (P < 0.0001). CONCLUSIONS: The developed decellularized dental pulp matrix had pro-angiogenic properties characterized by the retention of native vasculature and angiogenic growth factors. Seeding of hDPSCs into the DDP led to progressive repopulation of the vasculature, enhanced expression of markers involved in angiogenesis in hDPSCs and improved in vivo vascularization capacity. The se suggest that a combination of DDP and hDPSCs have the potential to provide a promising vascularization promoting strategy for dental pulp regeneration.

European Society of Endodontology position statement: Surgical extrusion, intentional replantation and tooth autotransplantation: European Society of Endodontology developed by.

This European Society of Endodontology (ESE) position statement on surgical extrusion, intentional replantation and tooth autotransplantation represents the consensus of an expert committee, convened by the ESE. A narrative review in the International Endodontic Journal (Plotino et al. 2020, international Endodontic Journal, 53, 1636-52) formed the basis for the position statement. The review provided detailed information on the background, clinical procedures and the outcome of surgical extrusion, intentional replantation and tooth autotransplantation techniques. The aim of the current statement is to summarize the best available evidence on these clinical techniques to provide appropriate clinical guidance to undergraduate and postgraduate students, dental practitioners, clinical teachers and researchers. The current position statement will be updated by the ESE periodically to reflect new evidence as it becomes available to provide the most current treatment guidance for clinical practice.

Clinical procedures and outcome of surgical extrusion, intentional replantation and tooth autotransplantation - a narrative review.

Surgical extrusion is defined as the procedure in which the remaining tooth structure is repositioned at a more coronal/supragingival position in the same socket in which the tooth was located originally. Intentional replantation is defined as the deliberate extraction of a tooth and after evaluation of root surfaces, endodontic manipulation and repair, placement of the tooth back into its original position. Tooth autotransplantation is defined as the transplantation of an unerupted or erupted tooth in the same individual, from one site to another extraction site or a new surgically prepared socket. The advent of titanium implant rehabilitation has reduced the use of these treatments in day-by-day clinical practice; however, the re-emerging trend to conserve and preserve natural sound tissues has led to a rediscovery of these treatments. All three distinct surgical methods are closely related, as they act to treat teeth that cannot be predictably treated using other more conventional procedures in endodontics, periodontics and restorative dentistry. Furthermore, these procedures share the same treatment approach and include the atraumatic extraction of a tooth, visual inspection of the tooth/root and its subsequent replantation. The clinical procedures for surgical extrusion, intentional replantation and tooth autotransplantation treatment have undergone several changes in recent years, and currently, there are no clear clinical treatment protocols/guidelines available. The clinician should be aware of the outcome of these treatments. Hence, the aim of this narrative review is to provide the background, clinical procedures and outcomes of surgical extrusion, intentional replantation and tooth autotransplantation.

Management of discolouration following revitalization endodontic procedures: A case series.

AIM: To discuss the causes and management of tooth crown discolouration following regenerative/revitalization endodontic therapy (RET) and to present the decision-making process affecting three RET-related cases of crown discolouration. SUMMARY: This case series outlines several approaches used in the management of post-RET discolouration. The cases highlight the influence of the material used in creating a coronal seal on the choice and success of the management techniques provided. The first two cases highlight successful management of post-RET discolouration through the use of internal-external bleaching. The use of internal bleaching in the third case was not recommended; therefore, external bleaching followed by a direct composite veneer was carried out.

A prospective clinical study of regenerative endodontic treatment of traumatized immature teeth with necrotic pulps using bi-antibiotic paste.

AIM: To evaluate the treatment outcomes of a revitalization endodontic technique (RET) for the management of traumatized immature teeth with necrotic pulps in children. METHODOLOGY: Fifteen healthy children (age range = 7-10 years) with traumatized immature maxillary incisors with necrotic pulps treated with bi-antibiotic revitalization endodontic technique were prospectively assessed over approximately two years (mean = 22 months). One operator undertook all treatments, clinical reviews and standardized radiographic exposures with radiographic analysis being carried out by two calibrated experienced clinicians. Crown colour change was assessed using an objective published methodology. Wilcoxon signed-rank test was used to compare root lengths, root dentinal widths and apical foramen widths over time. RESULTS: Interoperator measurement reliability was consistently strong for all measurements. There was no significant difference in root lengths or root dentinal wall widths following RET. A significant difference in apical foramen widths was observed after 2 years (P = 0.013) with resolution of clinical signs of infection in all cases. Despite omitting minocycline and using Portland cement (nonbismuth containing cement), a noticeable crown colour change (yellower, redder and lighter), as measured by an objective colour measurement system with ΔE = 7.39, was recorded. Most patients, however, were satisfied with the aesthetic outcome. CONCLUSION: Traumatized immature teeth with necrotic pulps treated with revitalization endodontic technique did not demonstrate continuation of root development or dentine formation when assessed by periapical radiographs. However, apical closure and periodontal healing were observed. A measurable change in crown colour (yellower, redder and lighter), with mostly no aesthetic concern to the patients/parents, was also observed.

A biocompatible decellularized pulp scaffold for regenerative endodontics.

AIM: To investigate the feasibility of decellularizing the entire dental pulp using a mild treatment protocol to develop a decellularized biological extracellular matrix scaffold for use in regenerative endodontic procedures. METHODOLOGY: Decellularized human dental pulps were assessed using histological and immunohistochemical methods, scanning electron microscope and DNA quantification assay. Cytotoxicity assays to determine decellularized scaffold biocompatibility were also performed. Decellularized scaffolds were seeded with human dental pulp stem cells and cell viability assessed using Live/Dead® stain. Quantitative data were analysed statistically using Student's t-test and one-way analysis of variance to compare mean values between groups depending on group numbers. RESULTS: Assessment of decellularized tissues revealed an acellular matrix with preservation of native tissue histoarchitecture and composition. Decellularized tissues showed no evidence of cytotoxicity, with cell growth in direct contact with the scaffold and no reduction in cellular activity following extract incubation. Furthermore, the scaffold was able to support human dental pulp stem cell viability and attachment following recellularization. CONCLUSIONS: Promising results were observed in developing a decellularized biological scaffold derived from the dental pulp with the perseveration of extracellular structural components which are required for tissue-specific regeneration.

Comparing clinical outcomes in HIV-infected and uninfected older men hospitalized with community-acquired pneumonia.

OBJECTIVES: Outcomes of community-acquired pneumonia (CAP) among HIV-infected older adults are unclear. METHODS: Associations between HIV infection and three CAP outcomes (30-day mortality, readmission within 30 days post-discharge, and hospital length of stay [LOS]) were examined in the Veterans Aging Cohort Study (VACS) of male Veterans, age ≥ 50 years, hospitalized for CAP from 10/1/2002 through 08/31/2010. Associations between the VACS Index and CAP outcomes were assessed in multivariable models. RESULTS: Among 117 557 Veterans (36 922 HIV-infected and 80 635 uninfected), 1203 met our eligibility criteria. The 30-day mortality rate was 5.3%, the mean LOS was 7.3 days, and 13.2% were readmitted within 30 days of discharge. In unadjusted analyses, there were no significant differences between HIV-infected and uninfected participants regarding the three CAP outcomes (P > 0.2). A higher VACS Index was associated with increased 30-day mortality, readmission, and LOS in both HIV-infected and uninfected groups. Generic organ system components of the VACS Index were associated with adverse CAP outcomes; HIV-specific components were not. Among HIV-infected participants, those not on antiretroviral therapy (ART) had a higher 30-day mortality (HR 2.94 [95% CI 1.51, 5.72]; P = 0.002) and a longer LOS (slope 2.69 days [95% CI 0.65, 4.73]; P = 0.008), after accounting for VACS Index. Readmission was not associated with ART use (OR 1.12 [95% CI 0.62, 2.00] P = 0.714). CONCLUSION: Among HIV-infected and uninfected older adults hospitalized for CAP, organ system components of the VACS Index were associated with adverse CAP outcomes. Among HIV-infected individuals, ART was associated with decreased 30-day mortality and LOS.

The effect of varying concentrations of fluoridated milk on enamel remineralisation in vitro.

The aim of this in vitro single blind study was to investigate the dose response of fluoride in milk on enamel demineralisation and remineralisation under pH cycling using transverse microradiography (TMR). Enamel slabs (n = 11) with caries-like lesions were exposed to milk containing 6 different fluoride concentrations (0, 0.25, 0.5, 1.0, 5.0 and 10.0 ppm F). On each of the 14 days of the cycling period the lesions were exposed to five 2-minute periods of cariogenic challenge (1.5 mM CaCl2, 0.9 mM KH2PO4 and 50 mM acetic acid at pH 5.0) and two 5-min periods in milk plus 10 min in a milk/saliva (1:3) slurry. The slabs were stored at 37°C in artificial saliva throughout the cycling period and demineralisation and remineralisation was assessed by TMR using dedicated image software. Remineralisation (ΔZ) was observed in all fluoride groups in contrast to demineralisation in the non-fluoride control. Remineralisation was significant (p < 0.05) for all concentrations above 1.0 ppm F. The results showed that fluoride concentration in milk exhibited a clear dose dependency and that the presence of fluoride even at low concentrations promoted remineralisation in this pH-cycling model.

The design and development of surgical templates for premolar transplants in adolescents.

The aim of designing and fabricating the surgical templates was to assist the surgical component of premolar transplantation, by establishing and replicating the root dimensions of the donor premolar tooth. The correct template could be used to assess the socket preparation (width and depth) prior to placement of the transplant in the recipient site, obviating the need to repeatedly try the donor tooth for fit at the recipient site, thereby minimising trauma to the periodontal ligament of the donor tooth. Seventy-five mandibular premolars and 50 maxillary second premolars were selected with varying stages of root development. All teeth had been extracted for orthodontic reasons. The root was measured for its length (maxL) and maximum (maxW) and minimum width (minW) with digital callipers from standardised reference points. These measurements were then used in the design of premolar transplant templates. The mean measurements for second maxillary premolars were maxL 14.6 mm (± 1.7), maxW 8.3 mm (± 0.5) and minW 4.9 (± 0.3). The mean measurements for mandibular premolars were maxL 14.8 (± 1.6), maxW 7.4 (± 0.6) and minW 5.3 (± 0.5). From these measurements, a range of maxillary second premolar and mandibular premolar templates have been fabricated. These figures also inform the multidisciplinary planning process for the space requirements at the donor site prior to transplantation.

Best clinical practice guidance for treating deep carious lesions in primary teeth: an EAPD policy document.

PURPOSE: The European Academy of Paediatric Dentistry (EAPD) has developed this best clinical practice guidance to help clinicians manage deep carious lesions in primary teeth. METHODS: Three expert groups conducted systematic reviews of the relevant literature. The topics were: (1) conventional techniques (2) Minimal Intervention Dentistry (MID) and (3) materials. Workshops were held during the corresponding EAPD interim seminar in Oslo in April 2021. Several clinical based recommendations and statements were agreed upon, and gaps in our knowledge were identified. RESULTS: There is strong evidence that indirect pulp capping and pulpotomy techniques, and 38% Silver Diamine Fluoride are shown to be effective for the management of caries in the primary dentition. Due to the strict criteria, it is not possible to give clear recommendations on which materials are most appropriate for restoring primary teeth with deep carious lesions. Atraumatic Restorative Technique (ART) is not suitable for multi-surface caries, and Pre-formed Metal Crowns (PMCs) using the Hall technique reduce patient discomfort. GIC and RMGIC seem to be more favourable given the lower annual failure rate compared to HVGIC and MRGIC. Glass carbomer cannot be recommended due to inferior marginal adaptation and fractures. Compomers, hybrid composite resins and bulk-fill composite resins demonstrated similar values for annual failure rates. CONCLUSION: The management of deep carious lesions in primary teeth can be challenging and must consider the patient's compliance, operator skills, materials and costs. There is a clear need to increase the use of MID techniques in managing carious primary teeth as a mainstream rather than a compromise option.

Comparison of the effects of whole and juiced fruits and vegetables on enamel demineralisation in situ.

We compared the effect on enamel demineralisation in situ of both whole and juiced fruits and vegetables. Volunteers wore removable mandibular appliances carrying pre-demineralised human enamel slabs and consumed one of the test foods 7 times a day for 10 days. The test foods were apples, oranges, grapes, carrots, and tomatoes, consumed either whole (sugars located intrinsically) or as a juice (extrinsic or free sugars). Raisins containing 64% sugars, but intrinsic by definition, were also studied. The mineral profile of the enamel slabs was studied before and after the test period using transverse microradiography and showed further demineralisation for all test foods, irrespective of the form of consumption. Significant demineralisation was also observed with raisins. No significant differences were found between the solid and juiced foods. In conclusion, sugars present intrinsically on consumption had a similar demineralising potential as free sugars and could not be considered less cariogenic.

Evaluation of Mineral Trioxide Aggregate and Biodentine as pulpotomy agents in immature first permanent molars with carious pulp exposure: A randomised clinical trial.

AIM: There is insufficient evidence on the outcome of pulpotomies in carious exposed young permanent molars with newer biomaterials. This study aimed to compare Mineral Trioxide Aggregate (MTA) and Biodentine as pulpotomy materials in carious exposed vital immature mandibular first permanent molars. MATERIALS AND METHODS: Study design: Sixty immature first mandibular permanent molars, with carious exposure were randomly assigned to an MTA or Biodentine group in a split-mouth design. After the amputation of the coronal pulp, the pulp stumps were covered with one of the study materials and coronal restorations placed. Blinded clinical and radiographic evaluations were performed at baseline. Following this there were evaluations at 6, 12 and 18 months where comparisons between and within the two groups were made. RESULTS: A high success was observed in both groups for all outcome measures for clinical success, with no significant difference between them. The mean survival time for the Biodentine and MTA groups was (17.8 and 18 months) with 95% confidence interval (17.4-18.2) and (18 .0-18.0) months respectively. Similarly, there were no significant differences between the Biodentine and MTA groups for radiographic success (P<0.001) with an increase in root length and increasing apical closure observed in both groups. CONCLUSIONS: Both materials were equally effective in the treatment of cariously exposed vital immature mandibular first permanent molars.

Rinsing with water for 1 min after milk formula increases plaque pH.

PURPOSE: Parents find it challenging to follow recommendations to brush young children after feeds at night despite the increase caries risk. This study compared three clinical recommendations (dilution, rinsing and wiping) on plaque pH after formula consumption. METHODS: Eighteen subjects were recruited. The five interventions with 2-week washout between visits included: Rinse with undiluted formula for 30 s (UF); rinse with 50% diluted formula for 30 s (DF); rinse with undiluted formula for 30 s, followed by rinsing with water for 1 minute (UF/R); rinse with undiluted formula for 30 s, followed by wiping (UF/W); rinse with 10% sucrose for 30 s as control (C). Plaque samples were collected at baseline, 2, 5, 10, 20 and 30 min after the intervention and pH measured using the plaque sampling method. RESULTS: UF/R resulted in significantly smaller pH drops at 5, 10 and 20 min compared to UF. It also resulted in higher minimum pH (UF/R: 6.34 ± 0.36 Vs UF: 6.06 ± 0.40, p = 0.02), smaller maximum pH drop (UF/R: 0.63 ± 0.35 Vs UF: 0.90 ± 0.49, p = 0.03), smaller sum of change of hydrogen ion concentration (UF/R: 9.22 × 10-7 ± 7.8 × 10-7 Vs UF: 2.30 × 10-6 ± 2.6 × 10-6, p = 0.04), and smaller area under the curve (UF/R: 7.70 ± 5.44 Vs UF: 13.44 ± 9.44, p = 0.02). DF and UF/W did not result in any significant pH change compared to UF. CONCLUSIONS: Of the three clinical recommendations, only rinsing with water for 1 min after undiluted formula reduced plaque acidogenicity. Teeth wiping with a moist cloth and 50% dilution of infant formula did not have an effect on plaque acidity.

Revitalisation endodontic treatment of traumatised immature teeth: a prospective long-term clinical study.

PURPOSE: Continuation of root development following revitalisation endodontics (RET) has been shown to be unpredictable with lower success rates in traumatised teeth. This study reports the outcomes for RET in traumatised teeth over a review period of 4 years. METHODS: A prospective uncontrolled study, where RET was performed on traumatised upper immature anterior teeth with necrotic pulps in 15 children (mean age = 8.3 years), was conducted. Patients were reviewed at 3, 9, 12, 24, and 48 months, where clinical and radiographic assessments were performed. At the last review appointment, patients and parents answered questions assessing their perception and acceptance of tooth colour change over time. McNemar's Exact test and linear mixed model assessment were used to assess changes in pulpal electrical response and radiographic evidence of continuation of root development over time, respectively. RESULTS: There was 83.3% healing with no significant changes in EPT responses, and no significant changes in root lengths, while significant changes in root widths (p < 0.05) and root apex widths (p < 0.001) were found over time. Twenty-five percent of patients and 33% of parents felt that there were changes in tooth colour following RET over time. CONCLUSION: Within the limitations of this study, traumatised teeth treated using RET showed no significant root lengthening, however, acceptable periapical healing, slow thickening of root dentinal walls, and rapid development of apical closure were evident over a period of 43 months. Using Portland cement and omitting minocycline, did not eliminate crown colour change following RET.

Best clinical practice guidance for prescribing dental radiographs in children and adolescents: an EAPD policy document.

BACKGROUND: The European Academy of Paediatric Dentistry (EAPD) proposes this best clinical practice guidance to help practitioners decide when and how to prescribe dental radiographs in children and adolescents. METHODS: Four expert working groups conducted each a systematic review of the literature. The main subjects were radiation protection, intraoral dental radiography (bitewing and periapical radiographs), panoramic radiography (PR) and cone-beam computed tomography (CBCT). In addition, three workshops were held during the corresponding EAPD Interim Seminar in Chania (Crete, Greece) in 2019. On the basis of the identified evidence, all invited experts presented their findings and during the workshops aspects of clinical relevance were discussed. RESULTS: Several clinical-based recommendations and statements were agreed upon. CONCLUSION: There is no or low-grade evidence about the efficacy of dental radiographic examinations in young populations. The given recommendations and rationales should be understood as best clinical practice guidance. It is essential to respect the radiological principles of an individualized and patient-specific justification. When a dental radiograph is required, its application needs to be optimized, aiming at limiting the patient's exposure to ionising radiation according to the ALADAIP principle (As Low As Diagnostically Achievable being Indication-oriented and Patient-specific).

Antibacterial properties and regenerative potential of Sr2+ and Ce3+ doped fluorapatites; a potential solution for peri-implantitis.

Scaffolds and implants in orthopaedics and regenerative dentistry usually fail because of bacterial infections. A promising solution would be the development of biomaterials with both significant regenerative potential and enhanced antibacterial activity. Working towards this direction, fluorapatite was synthesised and doped with Sr2+ and Ce3+ ions in order to tailor its properties. After experiments with four common bacteria (i.e. E. Coli, S. Aureus, B. Subtilis, B. Cereus), it was found that the undoped and the Ce3+ doped fluorapatites present better antibacterial response than the Sr2+ doped material. The synthesised minerals were incorporated into chitosan scaffolds and tested with Dental Pulp Stem Cells (DPSCs) to check their regenerative potential. As was expected, the scaffolds containing Sr2+-doped fluorapatite, presented high osteoconductivity leading to the differentiation of the DPSCs into osteoblasts. Similar results were obtained for the Ce3+-doped material, since both the concentration of osteocalcin and the RUNX2 gene expression were considerably higher than that for the un-doped mineral. Overall, it was shown that doping with Ce3+ retains the good antibacterial profile of fluorapatite and enhances its regenerative potential, which makes it a promising option for dealing with conditions where healing of hard tissues is compromised by bacterial contamination.

A silver renaissance in dentistry.

AIMS: Recently, there has been interest in biological approaches for caries management, with techniques such as interim therapeutic restorations and Hall technique crowns showing comparative effectiveness against conventional restorative methods. One of these approaches is the use of silver diamine fluoride (SDF) as a restorative option. In this invited review, the aim was to critically appraise the currently available literature with regards to the indication, technique and outcome of SDF. METHODS: A review of the literature on SDF with a focus on its use in the primary dentition was conducted. RESULTS: There has been a recent explosion in the literature regarding the use of SDF, from in vitro studies on the mechanism to clinical studies on the effectiveness and patient acceptance of the technique. A systematic review in 2016 concluded that SDF application resulted in caries arrest in 81% of active lesions in primary teeth over 30 months. Additionally, over 60% of parents reported being satisfied with the aesthetic outcome with most children finding the procedure to be acceptable, comfortable and relatively pain-free. CONCLUSIONS: Silver diamine fluoride is a safe and effective alternative technique to caries management in the paediatric population. Given the ongoing worldwide debate on cost-effectiveness of biological approaches coupled with the increasing concerns and limited accessibility of treatment under general analgesia, SDF should form an important addition to every dentist's armamentarium.

Regenerative endodontic therapy for managing immature non-vital teeth: a national survey of UK paediatric dental specialists and trainees.

Background Several guidelines have been published advocating the use of regenerative endodontic therapy (RET) in managing non-vital immature permanent teeth. It is unclear, however, how many UK paediatric dental specialists routinely use this technique and their opinion of its outcomes, and barriers to its use.Aim To assess the knowledge, experience and the opinion of UK based paediatric dental specialists/trainees (UKPDS/T) and practitioners working in the capacity of paediatric dental specialists on the use of RET.Design A cross-sectional study using a 22-item questionnaire was developed using the Bristol Online Survey tool and circulated electronically to members of the British Society of Paediatric Dentistry between August and November 2016.Results Ninety-eight UKPDS/T completed the survey. A quarter of respondents (N = 24, 24.5%) reported using RET. Reasons cited for not using RET included lack of: training (N = 48, 45%), materials (N = 28, 26%), evidence (N = 17, 16%) and suitable cases (N = 6, 6%). Different protocols in terms of disinfection, medicaments, scaffolds, and obturation material were identified.Conclusions This survey highlights a low uptake of RET by current UKPDS and trainees with several barriers identified. Deviations from the current RET guidelines were identified. Recommendations addressing the use of RET in light of the findings of this survey were made.

The effect on dental enamel of the frequency of consumption of fluoridated milk with a cariogenic challenge in situ.

OBJECTIVES: To evaluate the effect on enamel of varying the frequency of consumption of 1.0 mg F in milk once per day, twice per day or once every other day under cariogenic challenge in situ. MATERIALS AND METHODS: In a controlled, randomised, cross-over, single-blind study, 25 subjects wore an intra-oral lower removable appliance with enamel slabs for 21 days during each study arm. Subjects used F-free toothpaste, the cariogenic challenge comprising of five 2 min dippings per day in a 12% sucrose solution. Subjects dipped the appliances in 50 ml of 5.0 ppm fluoridated milk for five minutes during the test period once per day, twice per day, and once every other day and drank 200 ml of the same milk, once per day, twice per day (100 ml each time), or once every other day (200 ml) immediately on re-inserting their appliance in order to replicate topical and systemic effects. Slabs were analysed with surface microhardness (SMH) for protection against further demineralisation and transverse microradiography (TMR) to assess changes in mineralisation. RESULTS: Using SMH, 200 ml of 5.0 ppm F milk once per day was significantly better than 100 ml of 5.0 ppm F twice/day (p < 0.05) and 200 ml once every other day, but not significantly. Using TMR there was a statistically significant difference in mineral loss of enamel between baseline and treatment for all groups, but not between groups. CONCLUSIONS: Drinking 200 ml of 5.0 ppm milk once per day every day protected enamel against further demineralisation whereas all three frequencies were effective in promoting remineralisation.