Improving Oral Health with Fluoride-Free Calcium-Phosphate-Based Biomimetic Toothpastes: An Update of the Clinical Evidence

As the demand for clinically effective fluoride-free oral care products for consumers increases, it is important to document which types of toothpastes have been shown in clinical studies to be effective in improving oral health. In this review, we included different indications, i.e., caries prevention, improving periodontal health, reducing dentin hypersensitivity, protecting against dental erosion, and safely improving tooth whitening in defining what constitutes improvement in oral health. While there are several professional and consumer fluoride-containing formulations fortified with calcium-phosphate-based ingredients, this review focuses on fluoride-free toothpastes containing biomimetic calcium-phosphate-based molecules as the primary active ingredients. Several databases were searched, and only clinical trials in human subjects were included; in vitro and animal studies were excluded. There were 62 oral health clinical trials on biomimetic hydroxyapatite (HAP), 57 on casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), 26 on calcium sodium phosphosilicate (CSPS, or so called Bioglass), and 2 on β-tricalcium phosphate (β-TCP). HAP formulations were tested the most in clinical trials for benefits in preventing caries, dentin hypersensitivity, improving periodontal health, and tooth whitening. Based on the current clinical evidence to date, fluoride-free HAP toothpaste formulations are the most versatile of the calcium phosphate active ingredients in toothpastes for improving oral health.


Introduction
Even in the 21st century, poor oral health remains a major human affliction burdening health care systems in countries all over the world. Dental decay (caries) is still the most common affliction of children and very common in adults [1]. Periodontal disease today is the main reason for tooth loss throughout industrialized countries [2]. However, these human afflictions are preventable with improved diets, healthy nutrition, and especially with improved oral hygiene using toothpastes with active ingredients designed to prevent these common health issues [3]. Furthermore, as teeth are expected to last for a lifetime in ageing populations, dental tissues need to be protected from dental erosion. Some oral care products help protect teeth from mineral loss improving the longevity of the dentition [4]. In addition, people today want whiter and healthier looking teeth. Adults value the cosmetic appearance of their teeth; a whiter dentition improves confidence, improves social acceptance and even employment prospects [5]. Therefore, there is a need to develop active ingredients for toothpastes designed to help with one or more of the preventive roles in home oral care.

Results
The results of the search are shown in Figure 1. A total of 144 clinical trials and in situ clinical studies resulted after applying the exclusion and inclusion criteria. The majority (>80%) of the clinical studies were conducted on HAP-and CPP-ACP-containing toothpastes. Clinical studies on CSPS were mostly on dentin hypersensitivity (DH), and there were only two clinical trials found testing fluoridefree TCP toothpaste. With so many search term combinations, the Google Scholar search yielded an imprecise and excessively large number of titles which, after rapid screening, contained many citations, duplicates, and irrelevant publications. The focus was, therefore, on the titles retrieved in the PubMed, Scopus, and Web of Science databases. Both Scopus and Web of Science permitted "search within results" where subsets of publications were obtained from the large list of publications found using the starting primary search word (e.g., "hydroxyapatite"). of publications were obtained from the large list of publications found using the starting primary search word (e.g., "hydroxyapatite").  Table S1 shows the distribution of the clinical studies found using the main databases as a result of the various combinations of search terms. The publications that were retrieved in full and carefully read for each of the calcium-phosphate-based toothpaste active ingredients are summarized in Tables 1-4. Some studies were cited more than once in the tables because they examined more than one aspect of improving oral health in the same study.     Table S1 shows the distribution of the clinical studies found using the main databases as a result of the various combinations of search terms. The publications that were retrieved in full and carefully read for each of the calcium-phosphate-based toothpaste active ingredients are summarized in Tables 1-4. Some studies were cited more than once in the tables because they examined more than one aspect of improving oral health in the same study.
No studies were found.

Hydroxyapatite (HAP)
The authors of this review have previously published systematic reviews of the clinical evidence that HAP reduces dental caries [26], reduces dentin hypsersenstivity [27], and improves tooth color [5]. That literature has been updated in this review to include the most recent publications. A total of 62 clinical trials were found where HAP toothpaste was shown to reduce caries, remineralize enamel and protect against erosion, reduce dentin hypesensitivity, improve tooth color, and support gingival health (Table 1).

Casein Phosphopeptide-Amorphous Calcium Phosphate (CPP-ACP)
A total of 57 clinical trials were found on CPP-ACP toothpaste showing that this form of calcium-phosphate-based toothpaste reverses white spot lesions, protects against dental erosion and reduces dentin hypersensitivity ( Table 2). Only one study was found where CPP-ACP toothpaste was tested to improve gingival health. Several studies were found to show that CPP-ACP reduced dentin hypersensitivity in studies measuring the effectiveness of professional peroxide bleaching products and that the CPP-ACP did not interfere with the whitening process, but none were found where the active ingredient CPP-ACP was tested on its own in a toothpaste for whitening teeth.

Calcium Sodium Phosphosilicate (CSPS, Novamin, Biomin, Bioglass)
There have been several studies on fluoride toothpastes fortified with Novamin (CSPS), but those were not summarized in this review since the focus was on fluoride-free toothpastes. Recently, two studies examined CSPS as an active ingredient in fluoride-free toothpastes for controlling caries or white spot lesions [122,145]. There were 23 clinical studies found showing that CSPS was also capable of reducing dentin hypersensitivity. One study was found where CSPS as an isolated active ingredient was able to control gingival health. No studies were found where CSPS toothpastes were tested to improve the color of teeth. These studies are summarized in Table 3.

Beta-Tricalcium Phosphate (β-TCP)
The clinical literature on tricalcium phosphate toothpaste in improving oral health was very limited. While there were a number of in vitro studies and studies conducted on fluoride toothpaste with added TCP (called 'functionalized' TCP), only one clinical trial was found where a fluoride-free TCP toothpaste was tested in a clinical trial for reducing caries, and one clinical trial examined how fluorid-free TCP in toothpaste affected dentin hypersensitivity (Table 4).

Discussion
This systematic review was conducted to compare the clinical evidence that has been published on the calcium-phosphate-containing toothpastes designed to improve oral health. We were interested in comparing the calcium-phosphate-based active ingredients without fluoride. Many fluoride toothpaste formulations contain calcium phosphate additives in an attempt to improve the remineralization and protection of tooth enamel, but recent studies have shown that some ingredients, such as hydroxyapatite, perform as well if not better than fluoridated toothpaste [24,26,27]. Dental fluorosis has been an increasing concern, particularly in those countries that continue to fluoridate their drinking water supplies [169]. In addition, there are concerns that prenatal and even postnatal exposure to fluoride is linked to interference with brain function during early development and growth [170]. For these reasons, it is worthwhile to seek alternatives to fluoridated toothpaste.
The fluoride-free, calcium-phosphate-containing toothpaste formulations tested in the studies summarized in this review show great promise in that they have been shown in clinical trials to prevent dental decay, reverse white spot lesions, remineralize tooth enamel, protecting it from erosion, desensitize hypersensitive root surfaces and even improve gingival health, all while whitening and brightening the dentition.
There were 62 clinical studies found where HAP was the active ingredient and almost an equal number of clinical studies conducted on CPP-ACP. The vast majority of them used fluoride-toothpaste as positive controls. No study was conducted to compare HAP vs. CPP-ACP in a head-to-head clinical trial. Toothpastes containing CPP-ACP, which contains casein peptides, cannot be used in patients who are allergic to milk proteins. Neither can that toothpaste be given a 'vegan' designation. Calcium phosphate ingredients, if accidentally swallowed, are considered safe since they dissociate in the stomach into their constituent inorganic components (calcium and phosphate ions), which are not only harmless but actually contribute to needed dietary sources [171].
One other fluoride-free calcium-phosphate active ingredient that should have been considered but not included in the search was calcium glycerophosphate (CaGP), an active ingredient mentioned in the review by Enax et al. [172] on the remineralization strategies of molar incisor hypocalcification. While this ingredient is used mainly to fortify fluoride toothpaste, it has only been tested in three clinical trials as an active ingredient without fluoride [173][174][175]. In those recent trials, it has been shown to be effective on its own and should really be counted as the fifth active ingredient for fluoride-free calcium-phosphatecontaining toothpaste with the potential to reverse white spot lesions.

Future Directions
While the clinical evidence to date on the effectiveness of biomimetic fluoride-free calcium-phosphate ingredients in oral care products is already quite extensive and based on dozens of clinical trials, the development of new strategies and products for the prevention and control of oral diseases and maintaining good oral health should continue. Randomized clinical trials (RCTs) where calcium-phosphate-based toothpaste formulations are tested in head-to-head experiments have not been conducted. These would be useful in order to determine which active ingredients most meet the needs of the average consumer in improving overall oral health. Additional clinical trials are required using subjects in susceptible populations and in all age groups.

Conclusions
Because of the concern by families of the lasting negative effects of fluoride ingestion with the use of fluoridated toothpaste, there is increased interest by researchers in preventive dentistry to clinically test fluoride-free toothpastes for the potential to be effective in improving oral health. While there is extensive clinical evidence that the biomimetic approach of using hydroxyapatite, casein phopshopeptide-amorphous calcium phosphate, or calcium sodium phosphosilicate has proven successful, additional clinical studies would help identify the most effective active ingredients so that dentists can tailor targeted preventive regimens best suited for patients' needs. Based on the current clinical evidence to date, fluoride-free hydroxyapatite seems to be an all-round, versatile, and effective agent for improving oral health, in comparison to the other calcium phosphate active ingredients in toothpastes tested clinically.