**3.5 Local and systemic factors that influence tooth eruption**

Local factors which influence tooth eruption are ankylosed primary and permanent teeth, and ankylosis of primary molars with absence of permanent successors. Local factors generally cause delayed teeth eruption (Ertugrul et al., 2002). Referral to a pediatric dentist is recommended for appropriate treatment.

General factors associated with altered tooth eruption are Down syndrome (Trisomy 21 syndrome), cleidocranial dysplasia, congenital hypothyroidism (Cretinism), juvenile hypothyrodism, hypopituitarism, and achondroplastic dwarfism. Sporadic delayed eruption of teeth frequently occurs in children with Down syndrome. The first primary teeth may not appear until 2 years of age, and the dentition may not be complete until 5 years of age. The eruption generally follows an abnormal sequence and some of the primary teeth may be retained until 15 years of age (Jara et al., 1993; Ondarza et al., 1997). The prevalence and severity of early onset periodontal disease in children with Down syndrome are much

What is the Role of Pediatricians on Oral Health? 145

exclude sugar from the diet but rather to make the patient eat sugar in a sensible way sugar discipline (reasonable amounts and mainly at meal times). This also introduces the concept that it may be possible to substitute sucrose by substances which will impart sweetness but are not cariogenic, which is covered in more detail in section 4.3. However, a comparable study on human subjects will probably never be repeated as it would now be regarded as unethical to alter diets experimentally in directions likely to increase caries. Sugars integrated into the cellular structure of food (e.g. in fruit) are called *intrinsic sugars*. Sugars present in a free form (e.g. table sugar) or added to food (sweets, biscuits, etc.) are called *extrinsic sugars*. These are more readily available for metabolism by the oral bacteria. Therefore, they are potentially more cariogenic. Milk contains lactose. It is not generally regarded as cariogenic. Cheese and yoghurts, without added sugars, may also considered safe for teeth (Ahola et al., 2002). Bread, peanuts (not for children under 5 years), and sugarfree drinks are some examples of foods and drinks with low potential for dental caries. Thus the most damaging sugars for dental health are *non-milk extrinsic sugars (NMES)* (Moynihan, 2002). Both frequency and amount of sugars are associated with dental caries. However, at the level of the individual patient, it is more practical to advise limiting frequency of intake. Since frequency and amount of sugar consumed are closely associated and repeated sugar intakes mean several acid attacks on the teeth giving demineralization, efforts to reduce frequency should be made. Additionally, selecting food products that only lead a slight and/or short pH drop (above critical pH at which the enamel starts to dissolve around pH 5.5) is another measurement to reduce the unwanted local effects of diet with respect to caries. Many common food products containing fermentable carbohydrates can lead to a pH

Raw starch (e.g. raw vegetables) is of low cariogenity. Cooked and highly refined (e.g crisps) can cause caries, and combinations of cooked starch and sucrose (e.g. cakes, biscuits, sugared breakfast cereals) can be highly cariogenic. Fruits contain sugar (fructose, sucrose, and glucose) but fresh fruits appear to have low cariogenity. However, the same cannot be said for fruit juice. The juicing process releases the sugars from the whole fruit, and these drinks are potentially cariogenic. Dried fruits are also cariogenic since they are sticky, tending to adhere to teeth, and the drying process releases some of the intrinsic sugars (Moynihan, 2002). Some children have particular risk to caries because of dietary factors. These children should at least sound warning bells in the pediatrician's mind as well as

• Infants and toddlers provided with a feeding bottle at bedtime, or bottle hung up in the

• Children with an increased frequency of eating because of a medical problem, e.g.

• Children with an increased carbohydrate intake due to a medical problem, e.g. Chron's disease, chronic renal failure, other chronic illnesses, malnutrition, or failure to welfare • Children who are on medications causing reduced salivary flow, irradiation to the

• Children on long term and/or multiple medications: Are these sugar-based and/or do

of about 4 after their consumption.

region of salivary glands

they cause dry mouth?.

dentist's (Fejerskov & Kidd, 2003; Vadiakas, 2008):

• Infant and toddlers with prolonged breast-feeding on demand

cot for use during the night with a sugar-containing liquid

gastrointestinal disease, eating disorders, uncontrolled diabetes

higher compared to normal healthy and other mentally disabled children. A high prevalence of necrotizing ulcerative gingivitis is also reported (Carlstedt et al., 1996; Cichon et al., 1998). These higher prevalence relating periodontal diseases cannot only be explained by poor oral hygiene alone and may be the result of impaired immune responses and deficient phagocytic systems (Morinushi et al., 1997). Dental caries susceptibility is usually low in those with Down syndrome (Davidovich et al., 2010). Cleidocranial dysplasia (osteodentin dysplasia) has dental significance. The patients exhibit mandibular prognathism. The maxilla tends to be short vertically but not in sagittal way. The development of the dentition is delayed. One of the distinguishing characteristics is the presence of supernumerary teeth (Richardson & Deussen, 1994). The pediatric dentist serves as the coordinator of overall oral health care and disease prevention during an extended treatment regimen that usually includes two surgical interventions and three stages of orthodontic surgery. Hypothyroidism whether it occurs at birth or juvenile stage, causes delayed primary and permanent teeth eruption, and delayed primary teeth exfoliation (Mganga & Chindia, 1990). Hypopituitarism also causes delayed teeth eruption. Primary teeth may not undergo resorption but instead retained throughout the life of the patient. The underlying permanent teeth will develop but do not erupt (Conley et al., 1990). Delayed teeth eruption has been linked to other disorders, such as fibromatosis gingivae, chondroectodermal dysplasia, Gardner syndrome.

#### **4. Provision of a caries control measurements**

#### **4.1 Cariogenic diet (reduction in the intake of freely fermentable carbohydrates)**

Diet refers to the habitual allowance of food and drink taken by any person from day to day. Thus, the diet may exert an effect on caries locally in the mouth by reacting with the enamel surface and by serving as a substrate for cariogenic microorganisms. The food per se does not cause dental caries. Diet operates via the bacteria and the result may be a low pH at which the tooth starts to dissolve. The role of diet for proper tooth formation is a separate issue.

The consumption of sugar in substantial amounts is a recent trend in many areas of the world. Evidence linking caries and sugar has been evaluated from communities with low sugar consumption, and the results from severe dietary restrictions in many countries since 1940s. Another piece of evidence linking diet and caries concerns the rare hereditary disease fructose intolerance, which is caused by an inborn error of metabolism. Ingestion of foods containing fructose or sucrose causes severe nausea because of lack a certain liver enzyme. Consequently children with this disease avoid to eat these foods. The caries experience of them is considerably low, indicating that a group of children who are not able to tolerate many sugary foods are unlikely to develop much caries. There have been a number of noninterventional studies and animal experiments to relate dietary habits to the high prevalence of dental caries. According to a WHO study group, very little caries occurs in children when the national consumption level of sugar is below 10 kg per caput per annum (i.e., about 30 g/day), but a steep increase may occur from 15 kg upwards (WHO, 2003). Interventional human studies also designed in Sweden and Finland. Dentists now base much of dietary advice on the results of these two interventional studies, indicating that *the frequency of sugar intake should be reduced to confine sugar to meal times as much as possible.* The goal is not to

higher compared to normal healthy and other mentally disabled children. A high prevalence of necrotizing ulcerative gingivitis is also reported (Carlstedt et al., 1996; Cichon et al., 1998). These higher prevalence relating periodontal diseases cannot only be explained by poor oral hygiene alone and may be the result of impaired immune responses and deficient phagocytic systems (Morinushi et al., 1997). Dental caries susceptibility is usually low in those with Down syndrome (Davidovich et al., 2010). Cleidocranial dysplasia (osteodentin dysplasia) has dental significance. The patients exhibit mandibular prognathism. The maxilla tends to be short vertically but not in sagittal way. The development of the dentition is delayed. One of the distinguishing characteristics is the presence of supernumerary teeth (Richardson & Deussen, 1994). The pediatric dentist serves as the coordinator of overall oral health care and disease prevention during an extended treatment regimen that usually includes two surgical interventions and three stages of orthodontic surgery. Hypothyroidism whether it occurs at birth or juvenile stage, causes delayed primary and permanent teeth eruption, and delayed primary teeth exfoliation (Mganga & Chindia, 1990). Hypopituitarism also causes delayed teeth eruption. Primary teeth may not undergo resorption but instead retained throughout the life of the patient. The underlying permanent teeth will develop but do not erupt (Conley et al., 1990). Delayed teeth eruption has been linked to other disorders, such as fibromatosis gingivae, chondroectodermal dysplasia,

Gardner syndrome.

**4. Provision of a caries control measurements**

**4.1 Cariogenic diet (reduction in the intake of freely fermentable carbohydrates)**

tooth starts to dissolve. The role of diet for proper tooth formation is a separate issue.

Diet refers to the habitual allowance of food and drink taken by any person from day to day. Thus, the diet may exert an effect on caries locally in the mouth by reacting with the enamel surface and by serving as a substrate for cariogenic microorganisms. The food per se does not cause dental caries. Diet operates via the bacteria and the result may be a low pH at which the

The consumption of sugar in substantial amounts is a recent trend in many areas of the world. Evidence linking caries and sugar has been evaluated from communities with low sugar consumption, and the results from severe dietary restrictions in many countries since 1940s. Another piece of evidence linking diet and caries concerns the rare hereditary disease fructose intolerance, which is caused by an inborn error of metabolism. Ingestion of foods containing fructose or sucrose causes severe nausea because of lack a certain liver enzyme. Consequently children with this disease avoid to eat these foods. The caries experience of them is considerably low, indicating that a group of children who are not able to tolerate many sugary foods are unlikely to develop much caries. There have been a number of noninterventional studies and animal experiments to relate dietary habits to the high prevalence of dental caries. According to a WHO study group, very little caries occurs in children when the national consumption level of sugar is below 10 kg per caput per annum (i.e., about 30 g/day), but a steep increase may occur from 15 kg upwards (WHO, 2003). Interventional human studies also designed in Sweden and Finland. Dentists now base much of dietary advice on the results of these two interventional studies, indicating that *the frequency of sugar intake should be reduced to confine sugar to meal times as much as possible.* The goal is not to exclude sugar from the diet but rather to make the patient eat sugar in a sensible way sugar discipline (reasonable amounts and mainly at meal times). This also introduces the concept that it may be possible to substitute sucrose by substances which will impart sweetness but are not cariogenic, which is covered in more detail in section 4.3. However, a comparable study on human subjects will probably never be repeated as it would now be regarded as unethical to alter diets experimentally in directions likely to increase caries. Sugars integrated into the cellular structure of food (e.g. in fruit) are called *intrinsic sugars*. Sugars present in a free form (e.g. table sugar) or added to food (sweets, biscuits, etc.) are called *extrinsic sugars*. These are more readily available for metabolism by the oral bacteria. Therefore, they are potentially more cariogenic. Milk contains lactose. It is not generally regarded as cariogenic. Cheese and yoghurts, without added sugars, may also considered safe for teeth (Ahola et al., 2002). Bread, peanuts (not for children under 5 years), and sugarfree drinks are some examples of foods and drinks with low potential for dental caries. Thus the most damaging sugars for dental health are *non-milk extrinsic sugars (NMES)* (Moynihan, 2002). Both frequency and amount of sugars are associated with dental caries. However, at the level of the individual patient, it is more practical to advise limiting frequency of intake. Since frequency and amount of sugar consumed are closely associated and repeated sugar intakes mean several acid attacks on the teeth giving demineralization, efforts to reduce frequency should be made. Additionally, selecting food products that only lead a slight and/or short pH drop (above critical pH at which the enamel starts to dissolve around pH 5.5) is another measurement to reduce the unwanted local effects of diet with respect to caries. Many common food products containing fermentable carbohydrates can lead to a pH of about 4 after their consumption.

Raw starch (e.g. raw vegetables) is of low cariogenity. Cooked and highly refined (e.g crisps) can cause caries, and combinations of cooked starch and sucrose (e.g. cakes, biscuits, sugared breakfast cereals) can be highly cariogenic. Fruits contain sugar (fructose, sucrose, and glucose) but fresh fruits appear to have low cariogenity. However, the same cannot be said for fruit juice. The juicing process releases the sugars from the whole fruit, and these drinks are potentially cariogenic. Dried fruits are also cariogenic since they are sticky, tending to adhere to teeth, and the drying process releases some of the intrinsic sugars (Moynihan, 2002). Some children have particular risk to caries because of dietary factors. These children should at least sound warning bells in the pediatrician's mind as well as dentist's (Fejerskov & Kidd, 2003; Vadiakas, 2008):

What is the Role of Pediatricians on Oral Health? 147

result in some mild enamel fluorosis (McDonagh et al., 2000). The technical operation of water-fluoridation systems should be monitored and recorded regularly to prevent toxic

Fluoride toothpaste is the most widely used method of applying fluoride to teeth by so far. It is commonly used at home. It also has been using in community and school-based preventive programs. A recent systematic review concluded its beneficial use in preventing caries in children and adolescents, but only significantly for fluoride concentrations of 1000 ppm and above (Wong et al., 2011). Brushing teeth with fluoride containing toothpaste twice per day is recommended. The patients should brush before bed as the paste provides fluoride concentrations in saliva while the child is asleep. The effectiveness of fluoride toothpaste is concentration dependent. The relative caries preventive effects of fluoride toothpastes of different concentrations increase with higher fluoride concentration. The decision of what fluoride levels to use for children under 6 years should be balanced with the risk of fluorosis (Walsh et al., 2010). Studies have shown that use of fluoride toothpaste from an earl age is associated with higher levels of very mild fluorosis. The very mild grades are not aesthetically compromising, the use of fluoride toothpastes should continue to be promoted in communities, where or not they are served with fluoridated water or salt (WHO, 1994). However, in a recent study, it was indicated that there was weak unreliable evidence that starting the use of fluoride toothpaste in children aged <12 months may be associated with an increased risk of fluorosis. The evidence if use begins between the age of 12 and 24 months is equivocal. If the risk of fluorosis is of concern, the fluoride level of toothpaste for young children (under 6 years of age) is recommended to be lower than 1000 parts per million (ppm). More evidence from studies with low risk of bias is needed (Rasines, 2010). The author agrees with some other author's suggestion that children under 6 should use an adult-concentration paste (1000-1500 ppm fluoride), but a small pea-sized portion of it. The child should be encouraged to spit out excess paste and not swallow it. Children's pastes (500 ppm fluoride or less) could be recommended for children at low risk of caries living in an area where the water contains fluoride (Kidd, 2005). Some conclusions made by the WHO expert committee in 1994 are everyone should be encouraged to brush Daily with a fluoride toothpaste, every effort must be made to develop affordable fluoridated toothpastes for general use in developing countries, fluoridated toothpaste tubes should carry advice that for children under the age of 6 years brushing should be supervised and only a very small amount (less than 5 mm) should be placed on the brush or the

**4.2.3 Professionally applied fluoride (fluoride varnish in the pediatrician office)**

It has been previously reported that fluoride varnish (a concentrated form of sodium fluoride) reduced caries in the primary dentition by 33% and in the permanent dentition by 46% when compared with placebo (Morinho et al., 2011). Therefore, their wider use is encouraged by WHO (1994). Visits to primary care physicians and pediatricians are the norm during children's first few years of life in many countries. Surveys of pediatric primary care providers suggest that they are willing to provide preventive dental care for their pediatric patients (Pierce et al., 2002). Lewis et al found that 74 % of US pediatricians

effects of the element.

chewing-stick (WHO, 1994).

**4.2.2 Fluoride-containing toothpaste**

Measures to reduce caries risk and/or to stop ongoing caries activity in children are:

It is neither necessary nor practical to stop children eating sweets completely. On the contrary, children should be encouraged to eat a balanced meal before any sweets are given. Friend and relatives should be encouraged to bring gifts rather than sweets. The consumption of bed-time snack or drink (other than water) should be strongly discouraged, since salivary flow is virtually absent at night and plaque pH may remain low for many hours.

All dental professionals are encouraged to share current best practice oral health prevention strategies with their local community medical providers especially with pediatricians (Huston & Wood, 2009).

#### **4.2 Clinical use of fluorides**

There is substantial evidence that fluoride, through different applications and formulas, works to control caries development. The first observations of fluoride's effects on dental caries were linked to fluoride naturally present in the drinking water, and then from controlled water fluoridation programs. Other systemic methods to deliver fluoride were later suggested, including dietary fluoride supplements such as salt and milk. These systemic methods are now being questioned due to the fact that many studies have indicated that fluoride*'s action relies mainly on its post-eruptive effect from topical contact with the tooth structure. It is known that even the methods of delivering* fluoride *known as 'systemic' act mainly through a topical effect when they are in contact with the teeth.* The effectiveness of water fluoridation in many geographic areas is lower than in previous eras due to the widespread use of other fluoride modalities. Nevertheless, this evidence should not be interpreted as an indication that systemic methods are no longer relevant ways to deliver fluoride on an individual basis or for collective health programs. *Caution must be taken to avoid excess ingestion of* fluoride *when prescribing* dietary fluoride *supplements for children in order to minimize the risk of* dental *fluorosis, particularly if there are other relevant sources of* fluoride *intake - such as drinking water, salt or milk and/or dentifrice (*Sampaio et al., 2011; Jimenez-Farfan et al., 2011). Safe and effective doses of fluoride can be achieved when combining topical and systemic methods. Before considering supplementing fluoride, recommendations on how to avoid excessive fluoride intake should be followed (Buzalaf & Levy, 2011).

#### **4.2.1 Communal water fluoridation**

Studies evaluating the effect of water fluoridation on dental caries show a reduction in both the primary and permanent dentitions of about 50%. Community water fluoridation is safe and cost-effective and therefore, should be introduced and maintained wherever socially acceptable and feasible. The optimum fluoride concentration will normally be within the range 0.5-1.0 mg/l (ppm). At this currently accepted optimal level, water fluoridation will

• Sugars should be eliminated as fast as possible from the oral cavity. Foods needing

It is neither necessary nor practical to stop children eating sweets completely. On the contrary, children should be encouraged to eat a balanced meal before any sweets are given. Friend and relatives should be encouraged to bring gifts rather than sweets. The consumption of bed-time snack or drink (other than water) should be strongly discouraged, since salivary flow is virtually absent at night and plaque pH may remain low for many

All dental professionals are encouraged to share current best practice oral health prevention strategies with their local community medical providers especially with pediatricians

There is substantial evidence that fluoride, through different applications and formulas, works to control caries development. The first observations of fluoride's effects on dental caries were linked to fluoride naturally present in the drinking water, and then from controlled water fluoridation programs. Other systemic methods to deliver fluoride were later suggested, including dietary fluoride supplements such as salt and milk. These systemic methods are now being questioned due to the fact that many studies have indicated that fluoride*'s action relies mainly on its post-eruptive effect from topical contact with the tooth structure. It is known that even the methods of delivering* fluoride *known as 'systemic' act mainly through a topical effect when they are in contact with the teeth.* The effectiveness of water fluoridation in many geographic areas is lower than in previous eras due to the widespread use of other fluoride modalities. Nevertheless, this evidence should not be interpreted as an indication that systemic methods are no longer relevant ways to deliver fluoride on an individual basis or for collective health programs. *Caution must be taken to avoid excess ingestion of* fluoride *when prescribing* dietary fluoride *supplements for children in order to minimize the risk of* dental *fluorosis, particularly if there are other relevant sources of* fluoride *intake - such as drinking water, salt or milk and/or dentifrice (*Sampaio et al., 2011; Jimenez-Farfan et al., 2011). Safe and effective doses of fluoride can be achieved when combining topical and systemic methods. Before considering supplementing fluoride, recommendations on how to avoid excessive fluoride intake should be followed (Buzalaf &

Studies evaluating the effect of water fluoridation on dental caries show a reduction in both the primary and permanent dentitions of about 50%. Community water fluoridation is safe and cost-effective and therefore, should be introduced and maintained wherever socially acceptable and feasible. The optimum fluoride concentration will normally be within the range 0.5-1.0 mg/l (ppm). At this currently accepted optimal level, water fluoridation will

Measures to reduce caries risk and/or to stop ongoing caries activity in children are:

• Number of meals and snacks should be kept on a low level.

• Chewing lead to an increased salivation, which is desirable.

active

hours.

Levy, 2011).

**4.2.1 Communal water fluoridation**

(Huston & Wood, 2009).

**4.2 Clinical use of fluorides**

• Low sugar consumption is desirable from a cariological point of view

result in some mild enamel fluorosis (McDonagh et al., 2000). The technical operation of water-fluoridation systems should be monitored and recorded regularly to prevent toxic effects of the element.

#### **4.2.2 Fluoride-containing toothpaste**

Fluoride toothpaste is the most widely used method of applying fluoride to teeth by so far. It is commonly used at home. It also has been using in community and school-based preventive programs. A recent systematic review concluded its beneficial use in preventing caries in children and adolescents, but only significantly for fluoride concentrations of 1000 ppm and above (Wong et al., 2011). Brushing teeth with fluoride containing toothpaste twice per day is recommended. The patients should brush before bed as the paste provides fluoride concentrations in saliva while the child is asleep. The effectiveness of fluoride toothpaste is concentration dependent. The relative caries preventive effects of fluoride toothpastes of different concentrations increase with higher fluoride concentration. The decision of what fluoride levels to use for children under 6 years should be balanced with the risk of fluorosis (Walsh et al., 2010). Studies have shown that use of fluoride toothpaste from an earl age is associated with higher levels of very mild fluorosis. The very mild grades are not aesthetically compromising, the use of fluoride toothpastes should continue to be promoted in communities, where or not they are served with fluoridated water or salt (WHO, 1994). However, in a recent study, it was indicated that there was weak unreliable evidence that starting the use of fluoride toothpaste in children aged <12 months may be associated with an increased risk of fluorosis. The evidence if use begins between the age of 12 and 24 months is equivocal. If the risk of fluorosis is of concern, the fluoride level of toothpaste for young children (under 6 years of age) is recommended to be lower than 1000 parts per million (ppm). More evidence from studies with low risk of bias is needed (Rasines, 2010). The author agrees with some other author's suggestion that children under 6 should use an adult-concentration paste (1000-1500 ppm fluoride), but a small pea-sized portion of it. The child should be encouraged to spit out excess paste and not swallow it. Children's pastes (500 ppm fluoride or less) could be recommended for children at low risk of caries living in an area where the water contains fluoride (Kidd, 2005). Some conclusions made by the WHO expert committee in 1994 are everyone should be encouraged to brush Daily with a fluoride toothpaste, every effort must be made to develop affordable fluoridated toothpastes for general use in developing countries, fluoridated toothpaste tubes should carry advice that for children under the age of 6 years brushing should be supervised and only a very small amount (less than 5 mm) should be placed on the brush or the chewing-stick (WHO, 1994).

#### **4.2.3 Professionally applied fluoride (fluoride varnish in the pediatrician office)**

It has been previously reported that fluoride varnish (a concentrated form of sodium fluoride) reduced caries in the primary dentition by 33% and in the permanent dentition by 46% when compared with placebo (Morinho et al., 2011). Therefore, their wider use is encouraged by WHO (1994). Visits to primary care physicians and pediatricians are the norm during children's first few years of life in many countries. Surveys of pediatric primary care providers suggest that they are willing to provide preventive dental care for their pediatric patients (Pierce et al., 2002). Lewis et al found that 74 % of US pediatricians

What is the Role of Pediatricians on Oral Health? 149

programmes. Clinical studies have shown that xylitol, a natural, physiologic sugar alcohol of the pentitol type, can be used as a safe and effective caries-limiting sweetener. Habitual use of xylitol-containing food and oral hygiene adjuvants has been shown to reduce the growth of dental plaque, to interfere with the growth of caries-associated bacteria, to decrease the incidence of dental caries, and to be associated with remineralization of caries lesions. Other sugar alcohols that have been successfully used as sugar substitutes include D-glucitol (sorbitol), which, however, owing to its hexitol nature, normally has no strong effect on the mass and adhesiveness of bacterial plaque and on the growth of mutans streptococci. A tetritol-type alditol, erythritol, has shown potential as a non-cariogenic sugar substitute. Combinations of xylitol and erythritol may reduce the incidence of caries more effectively than either alditol alone (Makinen, 2011). Traditional delivering vehicles such as chewing-gums, hard candies and mints can only provide contact of the sugar substitutes with tooth surface for a few minute or even seconds. Therefore, novel delivery vehicles are still needed for the effective delivery of sugar substitutes before they can be considered as therapeutically effective.A group of peptides, known as casein phosphor peptide (CPP), have been shown to stabilize calcium and phosphate preserving them in an amorphous or soluble form known as amorphous calcium phosphate (ACP). Calcium and phosphate are essential components of enamel and dentine and form highly insoluble complexes, but in the presence of CPP they remain soluble and biologically available. This CPP-ACP complex applied to teeth by means of chewing-gum, toothpaste, lozenges, mouth rinses, or sprays is able to adhere to the dental biofilm and enamel hydroxyapatite providing bioavailable calcium and phosphate ions. When placed on the surface of a tooth with early carious lesions, pastes with CPP-ACP complexes can prevent tooth demineralization and improve enamel remineralization and enhance fluoride activity. Therefore, use of CPP-ACP based compounds offers a potential for use in the prevention of dental caries (Llena et al., 2009). Recently, probiotics have been investigating for dental caries prevention and inhibition. In caries, there are increases in acidogenic and acid-tolerating species such as mutans streptococci and lactobacilli, although other bacteria with similar properties can also be found and bifidobacteria, non-mutans streptococci, Actinomyces spp., Propionibacterium spp., Veillonella spp. and Atopobium spp. have also been implicated as significant in the aetiology of this disease (Aas et al., 2008). Therefore, to be able to develop probiotic or prebiotic interventions for applications in dental health care and to understand their mechanisms of action and potential risks, it is essential to have a clear understanding of the oral microbiota and their functions in dental/oral health and disease. However, some products have reached the market, there remains a paucity of clinical evidence to support the effectiveness of probiotics to prevent or treat caries (Meurman & Stomatova, 2007).

**4.4 Counseling families on basic oral hygiene**

Saied-Moallemi et al., 2008).

The considerable potential of mothers should be a major focus of (oral) health professionals in developing oral health promotion programs for children and adolescents. Several maternal cognitive, behavioral, and psychosocial factors were associated with young children's brushing practices. Oral health-specific self-efficacy and knowledge measures are potentially modifiable cognitions and intervening on these factors could help foster healthy dental habits and increase children's brushing frequency early in life (Finlayson et al., 2007;

expressed a willingness to apply fluoride varnish (Lewis et al., 2000). In promoting preventive dental health, pediatricians benefit all children and particularly the underserved. Therefore, pediatricians will require adequate training in oral health in medical school, residency, and in continuing education courses. It has been recently reported that *multiple applications of* fluoride *at the time of primary tooth emergence seem to be most beneficial to prevent dental caries formation*. Referrals to dentists for treatment of existing disease detected by physicians during regular visits limited the cumulative reductions in caries-related treatments, but also contributed to improved oral health (Pahel et al., 2011). Twice yearly application of fluoride varnish is indicated for the children over 6 years exposed to a greater cariogenic challenge or (in exceptional cases) when it is difficult to control caries in children under 6 years. Non-dental health care professionals should seek a professional advice from a pediatric dentist for appropriate application of the varnish.


**3. Hydrogels/MSCs scaffolds for tissue engineering**

The therapeutic effects of MSC transplantation largely attributed to the paracrine including soluble factors and extracellular vehicles (e.g., exosome and secretome), which could be rapidly sequestered and cleared [80]. Hydrogels are ideally suited for MSCs cultivation and are adequate to offer splendid delivery platforms, enhance vehicles retention rates and thus enhance immunomodulation and tissue regeneration after *in vivo* transplantation (**Figure 1**, **Table 1**) [80–82].


**4.2.6 AFLP (amplified fragment length polymorphism)**

Also termed infrequent restriction site PCR (IRS PCR). It, has been developed by Vos et al. (1995). L'AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters (i.e., linkers and indexers) to genomic restriction fragments followed by a PCR-based amplification with adapterspecific primers.

The optimal number of scorable bands (50–100) can easily be set by selection of the appropriate AFLP primers and restriction enzymes. These characteristics make AFLP a powerful fingerprinting technique which can be used in identification, epidemiology and taxonomy (Folkerstma et al. 1996; Huys et al. 1996; Janssen et al. 1996). In addition, the technique can be used to generate large numbers of molecular markers for linkage studies (Ballvora et al. 1995; Becker et al. 1995; van Eck et al. 1995).

condition (Erlich, 1989) in that only a single oligonucleotide of random sequence is

At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are

The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites. Recently, sequence characterised amplified regions (SCARs) analysis of RAPD polymorphisms (Bardakci & Skibinski, 1999) showed that one cause of RAPD polymorphisms is chromosomal rearrangements such as insertions/deletions. Therefore, amplification products from the same alleles in a heterozygote differ in length and will be detected as presence and absence

Although the RAPD method is relatively fast, cheap and easy to perform in comparison with other methods that have been used as DNA markers, the issue of reproducibility has been of much concern since the publication of the technique. In fact, ordinary PCR is also sensitive to changes in reaction conditions, but the RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility problem is usually the case for bands with lower intensity. The most important factor for reproducibility of the RAPD profile has been found to be the result of inadequately prepared template DNA (Welsh & McClelland, 1994). Differences between the template DNA concentration of 2 individuals'

Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer, DNA from virtually from all sources is amenable to amplification. Therefore, DNA from the genome in question may include contaminant DNA from infections and parasites in the material from which the DNA has been isolated. Special care is needed for keeping

Finally, due to the amplification conditions, RAPD method is sensitive to slight changes within amplification parameters, thus it is hard to achieve reproducibility. However, ribotyping is a

Also termed infrequent restriction site PCR (IRS PCR). It, has been developed by Vos et al. (1995). L'AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters (i.e., linkers and indexers) to genomic restriction fragments followed by a PCR-based amplification with

The optimal number of scorable bands (50–100) can easily be set by selection of the appropriate AFLP primers and restriction enzymes. These characteristics make AFLP a powerful fingerprinting technique which can be used in identification, epidemiology and taxonomy (Folkerstma et al. 1996; Huys et al. 1996; Janssen et al. 1996). In addition, the technique can be used to generate large numbers of molecular markers for linkage studies

DNA samples result in the loss or gain of some bands (Bardakci, 1996).

supplementary tool in conjunction with other typing methods (Yan et al., 2003).

out the DNA to be amplified from other sources of DNA.

**4.2.6 AFLP (amplified fragment length polymorphism)**

(Ballvora et al. 1995; Becker et al. 1995; van Eck et al. 1995).

employed and no prior knowledge of the genome subjected to analysis is required.

within an amplifiable distance of each other.

of bands in the RAPD profile.

adapterspecific primers.

For AFLP analysis, only a small amount of purified genomic DNA is needed; this is digested with two restriction enzymes, one with an average cutting frequency (like EcoRI) and a second one with a higher cutting frequency (like MseI or TaqI).

Double-stranded oligonucleotide adapters are designed in such a way that the initial restriction site is not restored after ligation, which allows simultaneous restriction and ligation, while religated fragments are cleaved again.

An aliquot is then subjected to two subsequent PCR amplifications under highly stringent conditions with adapter-specific primers that have at their 39 ends an extension of one to three nucleotides running into the unknown chromosomal restriction fragment.

An extension of one selective nucleotide amplifies 1 of 4 of the ligated fragments, whereas three selective nucleotides in both primers amplify 1 of 4,096 of the fragments. The PCR primer which spans the average-frequency restriction site is labeled.

After polyacrylamide gel electrophoresis a highly informative pattern of 40 to 200 bands is obtained. The patterns obtained from different strains are polymorphic due to (i) mutations in the restriction sites, (ii) mutations in the sequences adjacent to the restriction sites and complementary to the selective primer extensions, and (iii) insertions or deletions within the amplified fragments.

Optimization of restriction enzymes and adapter-specific primers is ongoing for the *Salmonella* (Garaizar et al., 2000), but the technique appears more reproducible than ribotyping techniques (Savelkoul et al., 1999). Some of the studies have shown specificity to the serotype level with occasional subserotype discrimination (Garaizar et al., 2000).

Alternative AFLP typing procedures are based on one enzyme with a single adapter and analysis by agarose gel electrophoresis (Gibson et al., 1998). A major improvement has been obtained using a fluorescent amplified fragment length polymorphisms (FAFLP) technique that followed the same principles of AFLP yet the adapter-specific primers were tagged with a fluorescent moiety (Tamada et al., 2001). Fluorescent tagged fragments are then accurately sized on an automated sequencer.

FAFLP analysis of *S. typhimurium* generated 45-50 fragments ranging in size from 80-430 bp, though only a subset of these fragments were polymorphic among the strains. FAFLP grouped the isolates into four distinct clusters while PFGE generated three clusters.

Sizing was enhanced by incorporation of a fluorescent internal marker (Tamada et al., 2001). This accurate sizing, combined with the ability to acquire and analyze the data as a gel image, electrophorogram or in a tabular data format will allow comparison of patterns among different laboratories or within databanks (Savelkoul et al., 1999).

FAFLP appears quite promising. Disadvantages include the need for a greater technical expertise. In fact, despite that AFLP has been considered as a highly discriminative method, it remains a labour- and cost-intensive technique (Riley, 2004). Set up costs may be prohibitive until automated sequencers become more affordable.


**3. Capsules**

In contrast to the glycocalyx, a bacterial capsule is a well-defined external structure with a characteristic composition for each bacterial genus and species that provides a number of advantages, which are primarily related virulence, to the producing microorganism.

One of the classic examples of the importance of the capsule is provided by *Streptococcus pneumoniae*, which is a gram-positive diplococci agent that causes pneumonia, meningitis and septicaemia. The virulent strains of this species produce a capsule that inhibits opsonisation and phagocytosis. The composition of the capsule is dependent on the producing bacteria. In the case of *Escherichia coli* K1, the capsule is formed by polysialic acid, in *S. pyogenes,* by hyaluronic acid and in *Streptococcus* group B by sialic acid. The biochemical composition of the capsules may be extremely diverse, which gives rise to a great amount of antigenic variability and presents a problem for the immunological mechanisms of the host in recognising these organisms [2].

It has been documented that the capsule participates in bacterial adhesion mechanisms and that its synthesis is stimulates by low stress conditions, such as the presence of serum, low Fe++ concentration and high CO2 tensions. Although in certain microorganisms, the presence of a capsule is discreet, in others, such as *Cryptococcus neoformans*, the capsule plays an essential role in the mechanisms for aggression against the host.


**2.1.1 Mediated contact interaction**

During haptic interaction mediated by a tool the subject holds a tool in his hand and the haptic information regarding the interaction is practically contained in the motion and the forces that are acting on the tool. An ideal device able to perfectly reproduce the haptic interaction can be equipped with a toll moke-up that is a faithful reproduction of the real tool. The problem of perfectly reproducing the tactile and kinesthetic sensory input is then reduced to the problem of imposing to such moke-up the same static and dynamic behavior of the real tool.

For this kind of interaction kinesthetic haptic interface are theoretically very efficient but in many cases their mechanical performance in terms of frequency response, fidelity in force reproduction and force resolution are still insufficient for high fidelity reproduction of the real feelings.

A lot of research efforts has been applied for improving and optimizing kinesthetic haptic interfaces specifically developed for mediated tool interaction and many systems are already commercialized and available as an off the shelf product.

For example, in the field of surgical simulators for training of laparoscopic surgery, catheter insertion, phlebotomy, or, in the domain of Virtual Prototyping and Assembly, mediated contact device have been successfully employed.


2.1.3. Findings by Fluoro-Jade B (FJB) Histofluorescence

In the vehicle-sham and ATX-sham groups, no FJB-stained cells (FJB-cells), which are degenerating or dead cells, were detected in CA1-3 (Figure 3(Aa,Ae,Ca,Ce)). In the vehicle-IR group, FJB-pyramidal cells in CA1-3 were observed from two days after IR (Figure 3(Ab,Ac,B,Cb,Cc,D)), and FJB-pyramidal cells of CA1-3 were dramatically increased at five days after IR; the number of FJB-pyramidal cells in CA1 and CA2/3 was 63 and 67 cells/300 μm2, respectively (Figure 3(Ad,B,Cd,D)). In the ATX-IR group, FJB-pyramidal cells in CA1-3 were not found until two days after IR (Figure 3(Af,Ag,B,Cf,Cg,D)). At five days after IR, many FJB-pyramidal cells were detected in CA1 and CA2/3 (26 and 13 cells/300 μm2, respectively), but the number was significantly low (41% in CA1 and 19% in CA2/3 of vehicle-IR group) when compared with the vehicle-IR group (Figure 3(Ah,B,Ch,D)). This finding means that the pyramidal cells in CA1-3 were lost (dead) after severe IR, and ATX could attenuate the death of pyramidal cells induced by severe IR.

**Figure 3.** (**A**,**C**) Representative photographs of FJB staining in CA1 (**A**) and CA2/3 (**C**) of the vehicletreated (left column) and ATX-treated (right column) groups in sham (**Aa**,**Ca**,**Ae**,**Ce**), and on day 1 (**Ab**,**Cb**,**Af**,**Cf**), day 2 (**Ac**,**Cc**,**Ag**,**Cg**), and day 5 (**Ad**,**Cd**,**Ah**,**Ch**) after IR. In the vehicle-IR group, FJB-pyramidal cells are detected in the SP of CA1-3 from two days after IR, and, at five days after IR, FJB-pyramidal cells (white asterisks in (**Ad**,**Cd**)) are significantly increased. However, in the ATX-IR group, decreased FJB-pyramidal cells (arrows in (**Ah**,**Ch**)) are observed in CA1-3 only at five days after IR. Scale bar = 60 μm; (**B**,**D**) Mean numbers of FJB- pyramidal cells in CA1 (**B**) and CA2/3 (**D**). The bars indicate the means ± SEM (*n* = 7, respectively; \* *p* < 0.05 vs. corresponding sham group, # *p* < 0.05 vs. vehicle-IR group).


*Article* **Does Relative Age Influence Organized Sport and Unorganized Physical Activity Participation in a Cohort of Adolescents?**

**Kristy L. Smith 1,\*, Mathieu Bélanger 2,3, Laura Chittle 1 , Jess C. Dixon 1 , Sean Horton 1 and Patricia L. Weir 1**

**Abstract:** Despite their prevalence, the longitudinal impacts of relative age effects (RAEs) on sport and other forms of physical activity (PA) are understudied. This study examined longitudinal participation patterns in organized sport (team and individual), unorganized PA, and non-participation with respect to RAEs in a prospective cohort of adolescents. Data from the first 24 cycles of the MATCH study were used for analyses. Elementary students (*n* = 929) were recruited from 17 schools in Atlantic Canada. Respondents self-reported PA three times/year. Mixed multilevel logistic models compared the likelihood of participating in each context across birth quarter. Chronological age and gender were considered, along with the interaction between chronological and relative age. Individuals born in Quarter 1/Quarter 2 were more likely to report participation in organized team sport but not individual sports. Relatively older participants born in Quarter 2 were more likely to report participation in unorganized PA. Increasing chronological age was associated with decreased participation in organized sport (particularly team-based) and increased non-participation. Gender was not associated with organized sport participation, but girls were under-represented in unorganized PA and more likely to report non-participation. The interaction parameters suggested that RAEs were consistent throughout adolescence in each context. Longitudinal analyses suggest RAEs are context dependent.

**Keywords:** relative age effects; participation trends; longitudinal; sport context; physical activity

#### **1. Introduction**

Despite efforts to communicate the importance of regular physical activity, high levels of physical inactivity remain a global concern [1]. Adolescence is a particularly concerning period as it is often marked by significant declines in physical activity participation [2–4]. "Physical activity" encompasses a variety of activities such as competitive sport, leisure walking, and occupational and domestic activities, among others [5,6]. Consequently, context is an important consideration when evaluating physical activity patterns [7]. Likewise, recommendations for increasing physical activity can incorporate different domains such as active transport (e.g., walking to school), participation in unorganized sport or active play [8], and even breaking up sedentary time with light intensity activity [9]. While participation in organized sport has been associated with a diverse range of positive outcomes among youth (e.g., enhanced resilience, character development, social skills [10,11]), the health benefits of physical activity (e.g., cardiorespiratory fitness, improved posture) might also be obtained in an unorganized context [12,13]. Thus, both organized and unorganized activity provide beneficial opportunities to increase participation. While the work to date by Bengoechea et al. [14] and Hardy et al. [15] present equivocal findings with respect to whether participation is higher in organized or unorganized activities, it is clear that

**Citation:** Smith, K.L.; Bélanger, M.; Chittle, L.; Dixon, J.C.; Horton, S.; Weir, P.L. Does Relative Age Influence Organized Sport and Unorganized Physical Activity Participation in a Cohort of Adolescents?. *Sports* **2022**, *10*, 97. https://doi.org/10.3390/sports 10070097

Academic Editors: Adam Leigh Kelly, Sergio L. Jiménez Sáiz, Sara Diana Leal dos Santos and Alberto Lorenzo Calvo

Received: 28 April 2022 Accepted: 21 June 2022 Published: 23 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

through adolescence, both categories of participation are important contributors to overall activity level [16].

One determinant that has been associated with participation in the physical activity context of organized sport is relative age. Relative age refers to the difference in age between individuals in a cohort [17,18], such as those grouped within the same grade in the school system. These age groupings are intended to help promote developmentally appropriate instruction and competition [19,20]. However, a one- or two-year age difference can result in significant physical, psychological, and experiential differences, particularly at young ages when a few months can represent a considerable proportion of a child's lived experience. In addition to experiential differences, age differences within a given age grouping can include considerable variability in biological maturity from childhood through to adolescence [18,21].

The term relative age effects (RAEs) refers to the (dis)advantages commonly associated with the aforementioned age groupings [22], providing a benefit to those who are relatively older (meaning that they were born closer to but following an arbitrary cut-off date) and disadvantaging those who are relatively younger in the same age group or cohort. With respect to sport, the advantage conveyed to those who are relatively older is assumed to be present when an over-representation of relatively older players is observed among sport participants (e.g., on a specific team), particularly at elite levels. While RAEs have predominantly been studied in team sports such as ice hockey, soccer, volleyball, and basketball (see Cobley et al. [23] for a review), individual sports, where success is predicted by strength or endurance capabilities, such as tennis [24], cross-country and alpine skiing [25], and sprinting [26,27], may also be affected. Potential consequences of RAEs include relatively younger players dropping out of organized sport at an earlier age [28–30] or adapting by taking up forms of activity that are less structured [31] or less competitive [32] in nature. To date, RAEs have primarily been found in male sport contexts ([23]), but a meta-analysis of RAEs in female sport shows a pervasive effect there as well (see Smith et al. [33]). Sex is nevertheless believed to be a significant moderator of the effect (Baker et al., 2010), with previous findings suggesting RAEs in females are more variable and weaker in magnitude when compared to males [23,34].

Several explanations and theoretical models of RAEs have been presented over the years. For instance, many relative age studies published to date have focused on maturation and selection (e.g., [20,23,35]). Being relatively older is often associated with greater physical and anthropometric maturity (e.g., height, weight, strength) [36]. During the maturational period, up to and including puberty, relatively older individuals may be viewed as more talented on the basis of their more advanced physical and anthropometric development. As a result, relatively older sport participants may benefit from a higher likelihood of being selected to elite teams, providing enhanced access to coaching and facilities, thereby facilitating greater skill development than their relatively younger peers.

Hancock et al. [37] argue that RAEs are largely tied to social influences. Specifically, RAEs are attributed to early experiences of success and the manner by which expectations of coaches and parents influence athletes' behaviors and self-perceptions, which ultimately impacts their level of achievement. Finally, a model proposed by Wattie et al. [38] suggests that the impact of relative age comes at the intersection of individual (e.g., gender, relative age), task (e.g., team or individual sport, organized vs. unorganized activity), and environmental constraints (e.g., age grouping policies) within developmental systems theory. Relative age can be viewed as one of many factors influencing the probability of sport participation, and there is opportunity for change throughout development. This developmental perspective is adopted within the current longitudinal study.

Relative age research has largely been cross-sectional in nature and focused on performance, examining the athlete population of a specific sport (e.g., basketball [39]) or sporting event (e.g., Olympic Games [40]). While this strategy has provided useful descriptions of the prevalence of RAEs, it does not inform about the potential longer-term effects of relative age advantages and/or disadvantages on sport and/or physical activity

participation for children and adolescents. Furthermore, evidence pertaining to RAEs in sport predominantly emerges from studies in organized sport and competitive contexts and is less commonly studied in recreational sport [23]. Observations from the few studies published to date suggest that RAEs may be highly variable within recreational contexts and influenced by a variety of factors and/or constraints [41–43]. Furthermore, published studies concerning the role of RAEs on outcomes associated with participation in unorganized sport and/or physical activity in children and adolescents are virtually non-existent.

Relative age effects have also been observed in a variety of areas within the school system such as physical education [44], high school leadership activities [45], standardized test scores (math and science [46]), university attendance [46], and emotional regulation [47]. These findings suggest that comparisons with peers occur in other contexts and could potentially impact the overall course of development for youth [48]. If relatively younger youth are habitually disadvantaged in organized activities and have fewer opportunities to benefit from sport system development, they could suffer from reduced perceptions of competence and eventually disengage from a physically active lifestyle. For example, perceived competence in a physical education setting has been positively associated with leisure-time physical activity (e.g., [49,50]). The impact of relative age may be evidenced in children's degrees of involvement not only in organized activities but also in unorganized activities or in a lack of participation altogether; these possibilities deserve further consideration.

To address the aforementioned gaps in the literature, the primary purpose of the current study is to examine the longitudinal patterns of participation in organized sport, unorganized physical activity, and non-participation with respect to relative age, in a prospective study of adolescents. The impact of gender and chronological age are considered, along with the interaction between chronological age and relative age within each participation/non-participation context. We hypothesize that a greater proportion of those who are relatively older participate in organized sport (both team and individual), while those who are relatively younger gravitate toward unorganized physical activities (where talent selection activities that favor the relatively oldest are notably absent), or a lack of participation altogether. Furthermore, it is expected that the hypothesized trends will become more pronounced with increasing chronological age for boys as they enter adolescence and become less pronounced for girls in alignment with previous meta-analytical findings [23,33].

#### **2. Materials and Methods**

#### *2.1. Respondents and Data Collection*

To accomplish the research objectives, the database for the Monitoring Activities of Teenagers to Comprehend their Habits (MATCH) study was queried to examine the same respondents in multiple activities rather than a single sport in isolation, which is a noted limitation of previous work. The MATCH study is an ongoing longitudinal prospective study in the province of New Brunswick, Canada. The complete study protocol is provided in Bélanger et al. [51]. Briefly, a convenience sample of 19 schools were recruited, and it included both French- and English-speaking students from a range of socioeconomic neighborhoods located in both rural and urban areas. Two of the 19 schools were excluded due to a low return rate of consent forms (below 50%). All grade five and six students from the remaining 17 schools were invited to participate in the study. Respondents ranged from 10 to 12 years of age at the time of recruitment, representing a critical timeframe when physical activity levels are often found to attain a peak before typically experiencing a decline as the transition from childhood to adolescence occurs [2,52]. Initial recruitment in the fall of 2011 included 802 of 1545 eligible students (response proportion of 52%), and other students from the participating schools were allowed to join the study in subsequent cycles (total number of respondents = 929). Please note within this study, the term 'respondent' refers to any individual who participated in the MATCH study, 'survey response' indicates

participation in a single MATCH data collection cycle, and 'participant' indicates reported participation in sport and/or physical activity (outlined further below).

The same respondents were asked to complete self-report questionnaires three times per year (fall, winter, and spring) at four-month intervals, which were combined to capture seasonal variations in one year of physical activity participation [6,51,53]. The first eight years of the MATCH study were used for the current analysis (*n* = 24 cycles). The questionnaires were completed under the supervision of a trained research assistant in a classroom setting, which was scheduled at a time that was convenient for the teacher. The initial session was 45–60 min in duration, and subsequent questionnaires were completed in 20–30 min. Respondents answered questionnaires in the first language of instruction at the school.

#### *2.2. Measures*

Respondents reported all forms of leisure time physical activity over the preceding four months from a list of 36 activities commonly practiced by youth in Atlantic Canada [54], which was similar to other validated physical activity checklists [55–57]. Respondents could also report additional activities under "other". The students were specifically instructed to disregard physical activity taking place during physical education classes because youth do not have control over the content of these classes [58]. The frequency of participation in each activity was collected using the response options "never", "once per month or less", "two to three times per month", "once per week", "two to three times per week", "four to five times per week", and "almost every day". Respondents were also asked to report "with whom" they had most often engaged in each activity by selecting from "by myself", "organized group or team", "sibling(s)", "friend(s)", or "parent(s)". The answer to this question was considered to be an indicator of the context in which each activity was most often practiced [59]. Respondents were described as 'participants' in the physical activity context being examined if they reported participating in any activity pertaining to each category "once per week" or more, similar to previous studies [14,60,61], thereby creating a binary measure (participant/non-participant).

For each of the cycles in the longitudinal sample, the classification of "relatively older" versus "relatively younger" must be made with reference to the cut-off date used to group individuals within the specific sporting activities. The dates used for activities recognized as "sports", defined by the existence of a recognized, provincial organization registered with Sport New Brunswick (SportNB), were obtained by contacting each respective organization listed in SportNB's online directory. If no response was received from the provincial organization, the national organization was contacted through the Sport Canada website. The majority of sports employed a 31 December cut-off date to group athletes. Other organized activities use cut-off dates as well, including the education system, which utilizes 31 December in the province of New Brunswick [62]. Therefore, the decision was made to code all respondents using this date because it is the most commonly encountered age grouping experienced by the respondents in this study. The coding of birth quarter proceeded as follows: Quarter 1—January to March; Quarter 2—April to June; Quarter 3—July to September; Quarter 4—October to December. Thus, the relatively oldest were identified as 'Quarter 1' subsequently followed by Quarter 2, Quarter 3, and Quarter 4 (i.e., the relatively youngest). This method of coding is consistent with previous studies (e.g., [63–65]).

Similar to other studies employing the MATCH sample, respondents were classified as "organized sport participants" if they reported participation with an "organized group or team" [61] in one of the recognized sports in New Brunswick. Organized sports were further categorized as "organized team sports" if the sport is most often practiced or played with more than one participant competing at the same time or multiple participants are a necessary element for competition. Alternatively, organized sports were classified as "organized individual sports" if participation most often occurs with only a single competitor competing at any given time. These definitions were consistent with previous

research [58,61]. Organized team sports that met the criteria outlined above included: ice hockey, ringette, baseball/softball, basketball, soccer, volleyball, and handball. Organized individual sports that met these criteria included: bicycling, track and field, jogging or running, badminton, tennis, downhill skiing or snowboarding, and cross-country skiing. The specific activity identified by the respondent was not important but rather the classification of "participant" in a particular context (i.e., organized team and/or individual sport).

To ensure accurate representation of each activity, sports that used a cut-off date other than 31 December were excluded to prevent bias from the use of an alternative date (i.e., 1 July for ice skating and 1 August for golf). Gymnastics was omitted because a reversal of the classic relative age pattern would be expected in this context. Late maturation is valued in aesthetic sports with peak performance occurring prior to the completion of maturation [38,66]. Accordingly, an over-representation of relatively younger participants and an under-representation of relatively older participants might be expected (i.e., reversal of the typical RAE pattern). This expectation was supported in the current data and therefore, gymnastics was removed from the organized sport analysis for this study to prevent an underestimation of the typical RAE present in this sample. Swimming was excluded because participants are categorized based on their chronological age on the day of competition. Other activities were excluded because the cut-off was unknown (i.e., football, boxing/wrestling), or multiple activities were grouped together (i.e., canoe/kayak and karate/judo/tai chi/taekwondo) in the questionnaire and could not be categorized consistently as an organized sport due to the absence of a provincial governing body for one or more of the activities.

Using the classification system derived by Ward et al. [61] and validated by MacKenzie and colleagues [67], five of the 36 activities were categorized as "unorganized physical activity", regardless of the context in which they were most often practiced, including trampoline, jump rope/skipping, games (e.g., chase, tag, hide and seek), home exercise, and weight training. Indoor chores and outdoor chores were excluded given the non-volitional and low intensity nature of these activities. Participation in the remaining activities was also considered to be unorganized if the individual reported to most often practice it alone, with friends, with siblings, or with a parent. A respondent who did not participate in any activity within a classification category was considered a "non-participant". A summary of the classifications is available in Appendix A.

#### *2.3. Data Analysis*

Prior to analyses, missing data for birth date (0.002%) was reviewed and corrected across all 24 cycles. A total of 12,061 survey responses were available for examination. Respondents (*n* = 929) participated in an average of 13 survey cycles each. Three objectives guided statistical analyses. First, the relative age trends in each of the five participation/nonparticipation contexts were assessed (i.e., any organized sport, organized team sport, organized individual sport, unorganized physical activity, and non-participation). Second, the impact of gender and chronological age were considered for each of the five contexts. Gender was self-reported by respondents in the MATCH study. In seven survey responses, (<0.01%) the respondent selected "other" as their gender category. These instances could not be included in the analyses due to insufficient sample size. Third, the longitudinal interaction between chronological age and relative age was considered within each participation/non-participation condition. To accomplish these objectives, a mixed multilevel logistic model was conducted for each of the five participation/non-participation contexts. The potential for intra-class correlation because of repeated measures among respondents and school-level clustering was accounted for by including random intercepts for these variables. Birth quarter (representing relative age), chronological age (by year – included as a continuous variable), and gender were included as independent variables, along with the interaction between chronological age and relative age. The fourth quarter (Quarter 4) was used as the comparison group. The interaction did not meet the criteria for statistical significance (*p* > 0.05) in any of the participation/non-participation contexts. Thus, the models presented in this paper do not include estimates for interaction parameters in the interest of parsimony.

#### **3. Results**

Our primary findings with respect to relative age trends, as indicated by the representation of participants in each birth quarter, are outlined below. Results are presented for organized sport (overall, team, and individual), unorganized physical activity, and nonparticipation, and they include findings for gender, chronological age, and the interaction between chronological age and relative age in each respective context. Of the participation contexts examined, unorganized PA had the most participants, and organized individual sport had the lowest number of participants throughout adolescence (Table 1). In general, there were more boys than girls reporting participation in all types of sport/PA.

**Table 1.** Average proportion of respondents in each context across the 24 survey cycles included in the analyses.

1 Respondents could be included in organized sport and unorganized physical activity (PA) simultaneously, but the non-participant category is exclusive. 2 Participants included in organized sport overall may have reported team and/or individual sport participation.

#### *3.1. Organized Sport*

In comparison to those born in Quarter 4, those born in Quarter 3 were less likely to report participation in any organized sport overall, *t* (976) = −2.205, *p* < 0.05 (Table 2a). However, when team and individual sport were analyzed separately, Quarter 1- and Quarter 2-born participants (i.e., the relatively oldest) were more likely to report participation in organized team sports when compared to Quarter 4 (i.e., the relatively youngest), *t* (1093) = 1.995, *p* < 0.05 and *t* (1003) = 2.138, *p* < 0.05, respectively (Table 2b). No statistical differences based on relative age were noted for organized individual sport participation suggesting equal representation of participants from each birth quarter (Table 2c).

Gender was not observed to be an important variable for any of the organized sport participation contexts (overall, team, or individual). Negative estimates for chronological age indicated that as participants became older, their participation in organized sports overall, *t* (915) = 1.356, *p* < 0.05, and organized team sports, *t* (3643) = −2.801, *p* < 0.01, declined but their reported participation in organized individual sport was maintained. The lack of statistical significance for any of the interaction parameters suggest that RAEs observed for organized sport participation overall, as well as for team and individual sport were consistent throughout adolescence (i.e., the same patterns of over-representation by birth quarter were observed across the years examined).

#### *3.2. Unorganized Physical Activity*

Participants born in the second quarter (i.e., relatively older) were found to be statistically over-represented compared to the fourth quarter (i.e., relatively younger) in unorganized physical activity participation, *t* (1544) = 2.731, *p* < 0.01 (Table 3).

**Table 2.** Odds ratios of participation in organized sport across relative age, (**a**) overall; (**b**) team; (**c**) individual.

1 Statistically significant values are indicated with bold text (*p* < 0.05) and \* (*p* < 0.01). 2 Birth quarter is identified by the following: Quarter 1 (Q1), Quarter 2 (Q2), Quarter 3 (Q3), and Quarter 4 (Q4).

**Table 3.** Odds ratios of participation in unorganized physical activity across relative age.

1 Statistically significant values are indicated with bold text (*p* < 0.05) and \* (*p* < 0.01). 2 Birth quarter is identified by the following: Quarter 1 (Q1), Quarter 2 (Q2), Quarter 3 (Q3), and Quarter 4 (Q4).

Participation in unorganized physical activity did not change with chronological age, but we noted that girls were less likely than boys to report participation in an unorganized context, *t* (1473) = −2.850, *p* < 0.01. The test of the interaction suggested that relative age trends (by year) were invariant in unorganized physical activity participation across the years examined.

#### *3.3. Non-Participation*

With respect to non-participation (i.e., individuals who did not report participation in an organized and/or unorganized context), Quarter 2-born individuals were found to be significantly under-represented compared to those born in Quarter 4, *t* (1556) = −3.374, *p* < 0.01 (Table 4).

**Table 4.** Odds ratios of non-participation across relative age.

1 Statistically significant values are indicated with bold text (*p* < 0.05) and \* (*p* < 0.01). 2 Birth quarter is identified by the following: Quarter 1 (Q1), Quarter 2 (Q2), Quarter 3 (Q3), and Quarter 4 (Q4).

Non-participation increased with chronological age, *t* (5216) = 2.657, *p* < 0.01, and there was an increased likelihood of non-participation among girls versus boys, *t* (1482) = 2.012, *p* < 0.05. The longitudinal trends (by year) with respect to relative age were stable in the non-participation category, as indicated by the lack of statistical significance in the test of the interaction.

#### **4. Discussion**

The longitudinal analyses undertaken in this study were based on eight years of data spanning late childhood to late adolescence. The primary purpose was to examine participation with respect to relative age in five participation/non-participation contexts. Secondary objectives included consideration of gender and chronological age as well as the interaction between chronological age and relative age. We found that relatively older (Quarter 1 and Quarter 2) respondents were more likely to report participation in organized team sport when compared to the relatively youngest (Quarter 4). This finding is consistent with past research and supports our hypotheses for the organized team sport context. However, in contrast to previous findings (e.g., [24–27,33]) and our hypotheses, no relative age trends were observed for organized individual sport participation. Furthermore, RAEs appeared to be associated with participation in unorganized physical activities, with Quarter 2-born participants over-represented. Notably, gender did not play a role in organized sport participation, but girls were under-represented in unorganized physical activities and more likely to report non-participation. Increasing chronological age was associated with decreased participation in organized sports overall and specifically in team-based contexts (but not individual sports); it was also associated with increased nonparticipation in this sample. Relative age effects appeared to be consistent throughout the developmental years examined in this study (i.e., the interaction did not meet the criteria for statistical significance (*p* > 0.05) in any of the participation/non-participation contexts).

An over-representation of relatively older participants in team-based sports supports the role of talent selection processes in perpetuating RAEs [23]. "Tryouts", whereby coaches and scouts scrutinize athletes' skills for the purpose of identifying the most talented individuals, are often required to gain membership on an organized team. These activities are believed to favor those who are relatively older, as they are likely to be taller/stronger and have acquired more life experience as a result of being chronologically older [18]. Once membership is gained, these selected individuals continue to benefit from being relatively older by having access to higher levels of training, coaching, and competition [20], resulting in an accumulated advantage from initial (albeit subtle) differences in growth, development, and experience [23,68].

Evidence for RAEs in individual sports has been less consistent in comparison to team sports [25]. Previous reports of RAEs have emerged from studies of individual sports for which physical attributes might provide an advantage such as tennis [24], cross-country and alpine skiing [25], and sprinting [26,27]. However, the examination of sport contexts that rely more predominantly on technical skills (vs. physical prowess) have not produced equivalent findings (e.g., golf [69], shooting sports [70]). It has been theorized that RAEs may be less prominent in individual sports due to comparisons between peers occurring

after performance, and they are often informed by more objective scores such as race time or a deductive rating system administered by a group of judges. In contrast, the peer-topeer comparisons associated with team selection processes are often made in the midst of competition or scrimmages where physical differences might be more apparent on the field of competition or during practice [25]. While "individual sport" in this study included several activities associated with physical demands, the lack of evidence for biased birth distributions suggests this type of sport context may be more likely to provide equitable opportunities for success. However, the compiled sample included a variety of sports, and each respective sport would need to be examined in detail to identify exact trends along with sport-specific factors contributing to the existence of RAEs or lack thereof. It is also important to note that several individual sport contexts were not available for examination, and competition/skill level was not considered in these analyses (i.e., a stronger risk of RAEs would be expected at elite levels of performance and may be somewhat diminished in a heterogenous sample) [23,33].

Gender (or sex, as reported in some relative age studies) has been identified as a significant moderator of RAEs in previous research, with male RAEs reported to be greater in magnitude versus the female effect [23], even when participation numbers are similar [34]. However, reported gender did not appear to modify trends observed in this longitudinal sample. This is not completely unexpected or incongruent with previous findings, given that RAEs were primarily associated with team-based sport in these analyses and likewise have been consistently identified in cross-sectional samples of both boys and girls in other team-based contexts (e.g., [23,33]). Furthermore, while the pattern of risk varies when samples are compared, RAEs are almost always present and expected to be present during the adolescent transition years, when variability in maturational differences is greatest [18,21].

An over-representation of Quarter 2-born participants (versus Quarter 4) was observed for unorganized physical activity participation. This finding may suggest that the impact of RAEs extends beyond the commonly examined context of organized sport, as talent selection processes would not be required to gain membership in these activities and engagement would be based on individual and/or parental volition. While direct comparisons for children and youth are not available in previous research for unorganized physical activity participation (Larouche et al. [71] reported findings for physical activity participation in a sample that included both adolescents and adults), this trend highlights the need to address developmental biases in both organized and unorganized contexts for this demographic given the importance of regular participation on health outcomes [12,72]. Reduced levels of cardiorespiratory fitness have been reported for the relatively youngest among children ages 9–12 years [73], and these findings may be associated with the extent to which these individuals feel confident about their ability to engage in various physical activity contexts.

An increased focus on the development of fundamental movement skills may support participation in unorganized forms of physical activity among the relatively youngest and could also support girls who were less likely to report participation in this context compared to boys. A future consideration is to examine whether basic movement skills (e.g., sprint, jump, throw, catch, balance) differ within a same-age cohort along with consideration of gender. Coincidentally, New Brunswick is one of the few Canadian provinces that require elementary schools to have a dedicated physical education specialist on staff [8], which could increase the likelihood that the development of basic movement skills is being promoted. These skills could potentially promote successful participation in a variety of physical activities across the lifespan and minimize impact of RAEs in an unorganized context [74,75]. It should also be noted that "unorganized" included a large number of activities (total of 36) in a variety of settings. Future studies should consider breaking this category down in order to identify any existing patterns in certain types of unorganized activity (e.g., group versus individual activities, domestic chores versus active transportation versus leisure pursuits).

"Non-participation" was defined as a lack of self-reported participation in an organized sport or unorganized physical activity at a minimum frequency of once per week, which is consistent with previous studies [14,60,61]. The relatively oldest participants born in Quarter 2 were less likely to report non-participation when compared to the relatively youngest born in Quarter 4. This further supports the notion that RAEs in organized contexts may potentially impact other aspects of health behavior during adolescence. Future work could examine whether this finding extends to sedentary behavior (e.g., screen time). An increased risk of non-participation was also observed for girls and with increasing chronological age. These findings are consistent with previous observations that activity levels begin to decline during adolescence (e.g., [76,77]) and should be taken into consideration when designing interventions to address non-participant behavior.

Returning to the theoretical framework of Wattie et al. [38], we have demonstrated that different constraints may be operating that impact adolescent participation in activities across a broad range of contexts. Individual constraints of chronological age and relative age continue to be present within the realm of organized sport overall, and specifically teambased activities, while relative age and gender impact less structured activities. Task-related constraints (e.g., team versus individual, organized versus unorganized) also differentiate participation. From a practical standpoint, the Wattie et al. framework and the findings of this longitudinal analysis (and future work) should be used to develop strategies to support the relatively youngest in various contexts. It may be necessary to address constraints at all levels (individual, task, and environmental) beyond the realm of organized sport. For instance, schools and/or community organizations may want to promote extracurricular opportunities by grouping students according to birth quarter to minimize the impact of age-related disparities. Children and youth could also be grouped according to their level of competency in a given activity. Furthermore, interventions are required to overcome RAEs throughout adolescence, since the relative age trends observed in this study were consistent across the eight years examined, suggesting that the relatively youngest did not overcome participation disadvantages at any point during the adolescent transition years.

The strengths of this study include that it is one of the first to provide a longitudinal examination of RAEs and it does so during the "pivotal" years of youth sport development [78]. Furthermore, the same respondents were evaluated in multiple contexts rather than in a single sport (e.g., team and individual organized sport, unorganized physical activity). The limitations of this study include those inherent in self-report questionnaires such as recall bias and social desirability bias, both of which could have resulted in misclassification and the over- or under-estimation of organized sport or unorganized physical activity participation. No objective measures of participation were utilized, and some organized sport contexts were not available for examination, or the cut-off date was unknown; however, the use of the questionnaire allowed for the investigation of several types of participation in addition to context. Furthermore, the study sample was not designed to be representative of a population, meaning that more research is needed to determine if the results of this study are generalizable to different youth populations [79].

#### **5. Conclusions**

Longitudinal analyses suggest RAEs are context dependent and persist through adolescence. The relatively oldest respondents (Quarter 1 and Quarter 2) were over-represented with respect to reported organized team sport participation, while individual sport contexts appeared to be unaffected, as evidenced by the lack of statistically significant differences in the birth distribution. Relative age inequities also appeared to be associated with participation in unorganized physical activities, with Quarter 2-born participants over-represented. Chronological age impacted organized sport participation overall and team-based contexts, while gender was observed to influence participation in unorganized physical activity and non-participation. Detailed research into specific constraints contributing to the observed patterns would be valuable.

**Author Contributions:** The data were collected by M.B., Principal Investigator of the MATCH study. K.L.S., M.B., L.C., J.C.D., S.H. and P.L.W. contributed to the conceptualization and design of the relative age analyses. K.L.S. prepared the data for statistical analyses, summarized the results, and drafted the manuscript with assistance from P.L.W. All authors assisted with review and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The data used in these analyses were drawn from the Monitoring Activities of Teenagers to Comprehend their Habits (MATCH) project, which was funded by the Social Sciences and Humanities Research Council (SSHRC, 435-2016-0888 and 435-2020-0809), by the SSHRC and Sport Canada joint Sport Participation Research Initiative (862-2010-0001 and 862-2014-0002), and by the New Brunswick Health Research Foundation (20130729). Support was also received through a SSHRC Doctoral Fellowship (K.L.S.). The funding sources had no direct involvement in the study design, in the collection, analysis, or interpretation of data, in writing of the report, or in the decision to submit the article for publication.

**Institutional Review Board Statement:** The MATCH study was reviewed and approved by the Comité d'Éthique de la Recherche du Centre Hospitalier de l'Université de Sherbrooke.

**Informed Consent Statement:** All respondents and their parents or guardians provided signed informed assent and consent to participate in the MATCH study, respectively.

**Data Availability Statement:** The original data analyzed for this study are available through a datasharing agreement with the MATCH study research team. More information on this may be obtained from the principal investigator of the MATCH study, Mathieu Bélanger.

**Acknowledgments:** The authors would like to thank Chris McCoy for assistance with the statistical analyses. The authors are also grateful to Joe Baker for his helpful comments in developing the framework for the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Table A1.** Adapted from Ward et al. [61].

1 Participation reported with an "organized group or team". 2 Participation reported with "sibling(s)", "friend(s)", "parent(s)/grandparent(s)", or "by myself".

#### **References**


*Article* **Perceived Consequences of Post-COVID-19 and Factors Associated with Low Life Satisfaction**

**Elisabeth Ekstrand 1,2,\*, Christina Brogårdh 1,3, Iben Axen 4, Agneta Malmgren Fänge 1, Kjerstin Stigmar 1 and Eva Ekvall Hansson 1,5**

**Abstract:** A significant number of individuals experience post-COVID-19 symptoms, but knowledge of perceived consequences and life satisfaction is lacking. Here, we investigate perceived consequences regarding everyday life, health, physical activity and work post-COVID-19 and factors associated with low life satisfaction. A total of 766 people (mean age 48; 672 women) experiencing post-COVID-19 symptoms at least two months after infection (mean 13 months) responded to an online survey. A majority (≥77%) perceived physical fatigue, mental fatigue, dizziness, reduced work ability, low life satisfaction and a reduced level of aerobic capacity. In the final logistic regression model (Nagelkerke R Square 0.296, *p* < 0.001), poor work ability was the most important factor for perceiving low satisfaction with life (Odds ratio 3.369, 95% CI 2.040–5.565, *p* < 0.001, Nagelkerke R Square 0.177). Reduced aerobic capacity, fatigue and living in a city also increased the odds of low life satisfaction. As people with post-COVID-19 report several long-term consequences, this suggests that there is a need for targeted care for this group. The results of this study can serve as guidance for healthcare authorities regarding important long-term consequences that should be considered in rehabilitation programs directed toward post-COVID-19.

**Keywords:** activities of daily living; COVID-19; health; life satisfaction; physical activity; post-COVID-19 condition; work


*2.1. Clinical Description of Studied Patients and of Their Families*

Comprehensive clinical, laboratory, and electrophysiological data of all index cases were collected. Two probands of them (P2, P4) also underwent cardiovascular magnetic resonance imaging (Siemens Trio scanner, Siemens Medical Solutions, Erlangen, Germany) as described previously [17]. All available relatives undertook cardiologic screening, which included physical examination, electrocardiography, and echocardiography as well as a collection of blood samples for genetic analysis. Patients with suspected disease-causing *DES* variants were subjected to a detailed neurologic assessment, measurement of serum creatine phosphokinase, nerve conductance, and electromyography of two muscles (left vastus medialis and left deltoid muscle), as reported previously [17].

The study was approved by the Institutional Review Board´s representing all clinical collaborators (Institute for Clinical and Experimental Medicine and Thomayer´s Hospital; 1st Faculty of Medicine of the Charles University and General Faculty Hospital; both Prague) and was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all probands.


**Acknowledgements**

The authors would like to acknowledge the laboratory interns and personnel which worked so diligently to support the research efforts of this and all other projects in our laboratory; Mr. Brandon Easparro, Ms. Taylor White, and Ms. Akelachi Okparanta. We would also like to thank Mr. Brent Barton for his contribution to our publications through graphics development, including but not limited to, the graphics used in this manuscript. Additionally, we would like to recognize the following researchers for their assistance in procuring the samples needed to conduct the research studies which allowed us to complete this chapter; Drs. Angel Rivera and Maria Nagy of Quorum X Diagnostics (Tucker, GA, USA), Dr. Michael Shannon of The Georgia Tech Research Institute (Atlanta, GA, USA), Dr. John Roback of Emory University School of Medicine (Atlanta, GA, USA), and Dr. Tonney Nyirenda of the University of Malawi College of Medicine (Blantyre, Malawi).


**Infiltration Characteristics and Spatiotemporal Distribution of Soil Moisture in Layered Soil under Vertical Tube Irrigation**

#### **Cheng Wang 1 , Dan Bai 1,\*, Yibo Li 1 , Xinduan Wang 2 , Zhen Pei 1 and Zuochao Dong 1**

Received: 31 July 2020; Accepted: 25 September 2020; Published: 29 September 2020

**Abstract:** The limited quantity of irrigation water in Xinjiang has hindered agricultural development in the region and water-saving irrigation technologies are crucial to addressing this water shortage. Vertical tube irrigation, a type of subsurface irrigation, is a new water-efficient technology. In this study, field and laboratory experiments were conducted to analyze (1) the infiltration characteristics and spatiotemporal distribution of moisture in layered soil and (2) the water-saving mechanism of vertical tube irrigation. In the field experiments, we analyzed jujube yield, irrigation water productivity (IWP), and soil moisture in the jujube root zone. In the laboratory irrigation experiments, two soil types (silty and sandy loam) were selected to investigate homogeneous and layered soil, respectively. Cumulative infiltration, wetting body, and soil water moisture distribution were also analyzed. Relative to surface drip irrigation, vertical tube irrigation resulted in slightly lower jujube yields but higher savings in water use (47–68%) and improved IWP. The laboratory experiments demonstrated that layered soil had less cumulative infiltration, a larger ellipsoid wetted body, slower vertical wetting front migration (hindered by layer interface), and faster horizontal wetting front migration than homogenous soil had. The irrigation amount for vertical tube irrigation decreased in layered soil, and water content increased at the layer interface. Vertical tube irrigation in layered soil facilitates the retention of water in the root zone, prevents deep leakage, reduces irrigation amount, and improves the IWP of jujube trees. This study aids the popularization and application of vertical tube irrigation technology.

**Keywords:** vertical tube irrigation; layered soil; soil moisture distribution; water-saving irrigation


*Edited by Vijay Singh Meena, Mahipal Choudhary, Ram Prakash Yadav and Sunita Kumari Meena*

*Sustainable Crop Production - Recent Advances* addresses various nutrient, crop, and soil management issues, including recent advances in sustainable food production in the context of the changing climate. Chapters present case studies on long-term field experiments in specific locations with a focus on the state of the art of sustainable agriculture production systems.

ISBN 978-1-80355-696-3

Sustainable Crop Production - Recent Advances

Published in London, UK © 2022 IntechOpen © Alex Holt / unsplash


**New Poly(Propylene Imine) Dendrimer Modified with Acridine and Its Cu(II) Complex: Synthesis, Characterization and Antimicrobial Activity**

**Paula Bosch 1,\*, Desislava Staneva 2, Evgenia Vasileva-Tonkova 3, Petar Grozdanov 3, Ivanka Nikolova 3, Rositsa Kukeva 4, Radostina Stoyanova 4 and Ivo Grabchev 5,\***

Received: 21 July 2019; Accepted: 16 September 2019; Published: 18 September 2019

**Abstract:** A second-generation poly(propylene imine) dendrimer modified with acridine and its Cu(II) complex have been synthesized for the first time. It has been found that two copper ions form complexes with the nitrogen atoms of the dendrimeric core by coordinate bonds. The new compounds have been characterized by nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), fourier-transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. The spectral characteristics of the modified dendrimer have been measured in different organic solvents, and a negative fluorescence solvatochromism has been observed. The antimicrobial activity of the dendrimers has been tested against model pathogenic microorganisms in agar and by broth dilution method. The cotton fabric treated with both dendrimers has been evaluated towards pathogenic microorganisms. The obtained modified cotton fabrics have been shown to hamper bacterial growth and to prevent biofilm formation. Dendrimer cytotoxicity has been investigated in vitro in the model HEp-2 cell line.

**Keywords:** dendrimer; metallodendrimer; acridine; antimicrobial activity; antibacterial cotton


*2.2. Muscles Dissection*

At the end of the protocols, the rats fasted for 12 h, the weight and glucose parameters of each group were measured to compare these values concerning each treatment. Subsequently, they were sacrificed for cervical dislocation; dissection was performed to obtain soleus and digitorum extensor longus (EDL) muscles of the two posterior extremities. The muscles of one of the extremities were maintained with Krebs-Ringer solution, to be later taken to isometric tension measurements. Simultaneously, the other extremity muscles were stored at −80 ◦C to be subsequently homogenized for biochemical tests. The Biuret method obtained the protein concentration of the homogenates [25].