The Physiology and Mechanisms of Growth

Introduction

Further elucidation of the physiologic processes involved in growth has been made in the past year. Not surprisingly, the role of nutrition has been further confirmed as one of the most important factors in growth. A selection of the most important articles published in the period from July 1, 2021 to June 30, 2022 dealing with physiology and mechanisms of growth is presented in this chapter.

In this year’s collection, several themes have been covered. Nutritional supplementation as a tool for optimizing growth has been further studied and could be used in everyday clinical practice in selected populations. Choosing the right protein and caloric value of the supplement, and also timing of the intervention, is paramount for the success. A link between nutritional state, puberty, and growth has been determined in MC3R, which is important not only for linear growth, but also for the management of chronic diseases and aging. Mechanisms behind the benefits of quality sleep in children for linear growth have been determined by studying the relationship between melatonin action and growth. Signaling processes involved in the regulation of endochondral ossification, as are matrix bound extracellular vesicles, have been described. In addition, studies in the growth plate showed the importance of local glucose metabolism. More candidates for potential manipulation in relation to increased growth in idiopathic short stature (ISS) have been identified. There are additional data on the effectiveness of growth hormone (GH) therapy in certain innate diseases of the growth plate or induced by glucocorticosteroid treatment. 

In the comments we try to explain why, in our opinion, these articles need to be especially highlighted. We, however, encourage the readers to read the full versions of the articles, when possible, and form their own opinions.

Different effects of soy and whey on linear bone growth and growth pattern in young male Sprague-Dawley rats

Comments: Which kind of protein is better for supporting linear growth? Should we choose plant-based (soy) or animal-based (whey) protein for this purpose? In this elegant study, it has been determined that although in the short term soy leads to a faster growth and better bone quality, in the long term whey was associated with better linear growth outcomes and better bone mineralization. The authors discuss the mechanisms behind the observed findings, as are protein amino acid composition of the supplements, calcium absorption, effects on the insulin-like growth factor 1 (IGF-1) circulating levels, and effects on microbiota. 
This study needs further validation in humans; however, it should be already considered in the planning of diets, especially in refeeding context. From the ecological point of view, the authors suggest that mixing plant-based and animal-based proteins in the human diet might be the most reasonable approach to a balanced and beneficial management plan. On the other hand, the timing of when soy- or wheybased products should be used is also important. Soy has a more rapid effect and whey has a more prolonged effect.

Effect of a nutritional supplementation on growth and body composition in short and lean preadolescent boys: a randomised, double-blind, placebo-controlled study

Comments: During puberty, there is an increased demand of the body for energy and nutrients. The nutritional needs during puberty are gender specific, with increased demand in boys. Therefore, suboptimal nutrition during puberty could result in suboptimal growth during this period, especially in boys who do not have sufficient energy intake. Effects of nutritional supplementation on growth were previously described [1, 2]. To this effect, an intervention with a specially designed formula was studied in a population of lean prepubertal boys in a double-blind randomized controlled study for 6 months. In comparison to the placebo, the formula had a higher caloric value and significantly increased levels of proteins, carbohydrates, and fat. In addition, it was fortified with calcium, iron, zinc, and vitamins A and C. Following the intervention, a significantly increased height was determined in boys receiving the formula. These data not only corroborate those related to the importance of nutrition in linear growth, but also suggest that they should be gender and pubertal stage specific. The described approach represents a new and validated management option in short and lean boys.

Associations of obesity with linear growth and puberty

Comments: Under- and overnutrition have an important influence on growth pattern and final height. This review is a must-read compilation of up-to-date studies on the effects of overnutrition/obesity on linear growth in children. Authors describe mechanisms leading to the described observations, which are important for the reader’s everyday clinical practice, when approaching children with obesity, as well as in planning preventive or curative measures for obesity. The authors emphasize the role of the adipose tissue, as an important endocrine organ in this respect. The role of leptin, possibly the main adipokine, is especially highlighted. Leptin affects linear growth by both directly acting at the growth plate and by regulating the GH-IGF-1 axis. In addition, the link between adipose tissue endocrine function and puberty start and progression and the effect this has on linear growth is discussed.

MC3R links nutritional state to childhood growth and the timing of puberty

Comments: The leptin-melanocortin pathway is possibly the most important regulator of energy intake and energy expenditure. Mutations in this signaling pathway are associated with increased appetite, development of obesity, and also other clinical features. Medications targeting this pathway are being developed to mediate this dysregulation. MC3R, which is mainly expressed in the brain, is a part of this system. Its role in the development of obesity is discussed; however, as this important work suggests it seems to be an important link between the timing of puberty, pattern of linear growth, and body composition. Namely, loss-of-function in this receptor has been linked to delayed puberty and reduced linear growth in addition to reduced lean mass and circulating IGF-1 levels. These findings further identify the leptin-melanocortin pathway as an important regulator of puberty and growth and suggest that signaling through MC3R might be responsible for selectively regulating puberty and growth. In addition, selectively stimulating the MC3R might preferentially lead to the development of lean mass (and not fat mass), which is of importance in chronic diseases management and aging.

Deletion of Glut1 in early postnatal cartilage reprograms chondrocytes toward enhanced glutamine oxidation

Comments: It has been recently determined that glucose transporter 1 (GLUT1)-mediated glucose metabolism has an essential role in embryonic cartilage development and linear bone growth. Lack of GLUT1 was linked to decreased osteoblast differentiation and mineralization [3,4].
GLUT1 seems to be the main glucose transporter in the cartilage. The role of glucose metabolism in postnatal cartilage growth was investigated in a mice model with selective early postnatal glucose transporter 1 (GLUT1) deletion in the chondrocytes. It was determined that GLUT1-mediated glucose metabolism is critical for postnatal growth plate development resulting in reduced linear bone growth. Mechanistically, it was linked to both by interfering with chondrocyte proliferation and matrix synthesis and processing. The results also show the metabolic plasticity of postnatal chondrocytes, since they are able to shift their metabolic pathways; in this case by using glutamine as an energy substrate, when glucose transport via GLUT1 was not possible. This shows the metabolic plasticity of the chondrocytes, which seems to be important when they are exposed to different metabolic demands during proliferation and differentiation and matrix synthesis and secretion. 
In addition, the results of the study show different responses to GLUT1 silencing in different parts of the cartilage system. In articulate cartilage chondrocytes, postnatal GLUT1 deletion results in diminished cellularity and loss of proteoglycans, which ultimately progress to cartilage fibrosis. These changes lead to earlier osteoarthritis. Altogether, this and related studies show the importance of glucose metabolism in growth plate chondrocytes both prenatally and in the early postnatal phase, the dysregulation leading to suboptimal growth.

Melatonin contributes to the hypertrophic differentiation of mesenchymal stem cell-derived chondrocytes via activation of the Wnt/β-catenin signalling pathway: melatonin promotes MSC-derived chondrocytes hypertrophy

Comments: In these 2 articles the role of circadian rhythms in endochondral ossification is described. Melatonin interacts with various types of stem cells and typically stimulates proliferation and transition to mature cell type. It has been shown that it has an especially important role in chondrogenesis and osteogenesis [5]. These 2 articles describe the mechanisms behind these observations. In the first article, it has been shown that melatonin mediates its action by periodically activating melatonin receptor 1, which then by downstream signaling pathways affects cell proliferation and matrix synthesis in the growth plate. In the second article, the direct role of melatonin in terminal differentiation of chondrocytes in endochondral ossification is described, by acting through the Wnt signaling pathway. These data further show the importance of central circadian rhythms in linear growth and identify novel pathways for possible manipulation in growth disorders.

LCN2 is a new diagnostic biomarker and potential therapeutic target in idiopathic short stature

Comments: In this interesting study, the authors have shown that serum LCN2 expression is upregulated in children with ISS. Serum LCN2 showed high sensitivity and specificity in discriminating children with ISS from GH deficiency, precocious puberty, and normal control individuals. LCN2 is an innate immune factor belonging to the lipocalin superfamily. 
It has been previously reported that LCN2 was overexpressed in patients with lupus nephritis and in other autoimmune inflammatory diseases. In an interesting set of studies, they have shown that overexpression of LCN2 suppresses food intake, and impairs chondrocytes proliferation and bone formation in the growth plate. The authors also showed in an animal study that bone growth impairment due to overexpression of LCN2 could be reversal when the overexpression stopped. Therefore, the authors concluded that LCN2 is a valid biomarker for ISS diagnosis and may be a potential target for ISS therapy.
These results are interesting and surprising. The diagnosis of ISS is made after all known causes of short stature are excluded in an intensive workup, which includes assessment of the GH-IGF-1 axis but also other known causes like celiac or other known inflammations like inflammatory bowel disease, by collecting detailed anamnestic story and laboratory workup including complete blood count, C-reactive protein, and blood chemical analysis. We have learned to believe that the diagnosis of ISS is a basket that includes different undiscovered causes of short stature. We know thatchildren with ISS might belong to a family where both parents and siblings are short (familial short stature) or an isolated case of short child in the family. I find it difficult to believe that all or even most children with ISS have a common cause. Therefore, I agree with the bottom-line of the recommendation made by the authors: a prospective, randomized, controlled, multicenter trial should be conducted to support or refute their findings.

Treatment of short stature in aggrecan-deficient patients with recombinant human growth hormone: 1-year response

Comments:  In this study, the authors demonstrated the results of 1 year of treatment of short stature in aggrecan-deficient patients. In 10 patients, the median height velocity increased from 5.2 cm/year before intervention to 8.3 cm/year after 1 year of GH treatment and increased the Height Standard Deviation Score (HtSDS) by +0.62. Interestingly, skeletal maturation did not advance inappropriately and no adverse events related to the therapy were observed.
I assume that for many years we were treating children with aggrecan deficiency thinking that we were treating children with ISS since the correct diagnosis was not identified. However, despite the fact that this study reports only 1 year of response to therapy of only 10 participants and with no control group, it is still an important description. More multicenter studies of that uncommon condition with appropriate comparative control for a longer duration are needed to sort out the full response to GH therapy in this group of short individuals.

Combined growth hormone and insulin-like growth factor-1 rescues growth retardation in glucocorticoid-treated mdxmice but does not prevent osteopenia

Comments: Duchenne muscular dystrophy (DMD) affects 1 in 4,000 live male births and is caused by mutations in the DMD gene on the X chromosome. The loss of dystrophin protein results in progressive replacement of muscle fibers by fat and fibrous tissue. Glucocorticoids are currently the mainstay of treatment of DMD. In this study the authors aimed to determine whether the combined administration of recombinant human growth hormone (rhGH) and IGF-1 could rescue the glucocorticoid-induced skeletal impairment and growth retardation in a mice model of DMD (mdx mice). The authors report that the combination of GH and IGF-1 increased somatic growth but did not improve the negative effects of long-term glucocorticoid treatment on bone growth or cortical bone development in their mice model. The question of how to overcome the effect of glucocorticoids on linear growth is not just limited to children with DMD. Many children are exposed to long-term glucocorticoid treatment for a variety of diseases with a deleterious effect on their longitudinal growth and bone metabolism. The use of animal models is the right way to go in testing possible medication that can change the course of growth in children who are exposed to long-term use of steroids despite the fact that animal models are not exactly equal in their response to therapy to those of human beings. I agree with the authors that more convincing evidence is needed before a clinical study is designed.

The role of matrix-bound extracellular vesicles in the regulation of endochondral bone formation

Comments: In this review, the authors update the readers on new information that was achieved in recent years related to the matrix-bound extracellular vesicles (MVs) in the regulation of endochondral bone formation. MVs were already reported in the literature in the 1960s. They are extracellular organelles ranging from 50 to 150 nm in diameter and are anchored to the extracellular matrix of the growth plate via integrin binding to collagen. They traditionally were known as the site of initial mineralization. With the progress made in laboratory methods, more information emerged. It became clear that in addition to enzymes and minerals and regulatory glycoproteins, MVs also contain micro-RNAs. It was also found that MVs, which are produced by the chondrocytes of the growth plate, have a different content of proteins and micro-RNAs if they are derived from the resting cells or from more mature cells like the pre-hypertrophic zone or hypertrophic cells. It was suggested that MVs have a role in transferring information between the cells within the growth plate. So, while the full role of the MV was not yet elucidated, more information is available to date and expected to come in the near future with the advancements of the laboratory research tool. This is a well-written review with beautiful figures which I recommend reading.

Effect of enteral zinc supplementation on growth and neurodevelopment of preterm infants: a systematic review and meta-analysis

Comments: Zinc, an important trace element in the human body, plays a critical role in linear growth during childhood via various mechanisms. Preterm infants, especially those who are very preterm, are at a high risk of zinc deficiency, as fetal zinc accretion occurs mostly after 24 weeks of gestation. Other factors include low body stores, renal and gastrointestinal losses, low zinc intake, and increased demands due to the relatively high growth rate in infancy. Zinc is also very important for protein synthesis. Recent nutritional approaches in very preterm infants include a high-energy and high-protein-based feeding regimen, which may necessitate greater amounts of zinc in the early days of a very preterm infant. Zinc deficiency in preterm infants is often subclinical but may be associated with growth, weight gain, and neurodevelopmental outcomes. The question on whether extra zinc supplementation can improve clinical outcome in preterm infants is unclear and current research has yielded conflicting results.
This is a systematic review and meta-analysis of randomized controlled trials of zinc supplementation in preterm infants, focusing on growth and neurodevelopmental outcomes. A total of 8 randomized controlled trials which included 742 preterm infants were reviewed. Seven of the studies reported growth parameters at 3–6 months corrected age; 1 study reported growth parameters just prior to discharge from the hospital. Three trials reported neurodevelopmental outcomes, with 2 studies reporting outcomes at 6–12 months corrected age, and 1 trial reporting outcome in less than 3 months corrected age. Results showed that zinc supplementation was associated with +0.5 SD higher in weight z-score (95% CI: +0.23 to +0.76) and +1.12 SD higher in length z-score (95% CI: +0.63 to +1.61). On the other hand, neurodevelopmental outcomes yielded less conclusive results. Most studies used a parent-reported questionnaire rather than objective assessments. The review identified that motor development may be better with zinc supplementation in preterm infants but not overall neurodevelopment. This is the first systematic review and meta-analysis of this topic and has raised important questions. An issue that was noted is the lack of safety data in the trials included and the relatively small number of cases included. Larger trials should be conducted to ascertain the safety of zinc supplementation and also address important issues like regimen, dose of zinc used, and timing of introduction as part of nutritional therapy.

Growth hormone treatment in the pre-transplant period is associated with superior outcome after paediatric kidney transplantation

Comments: Childhood chronic kidney disease is associated with poor growth leading to significant short stature, which is often disproportionate short stature with preferential involvement of the legs. Despite success with kidney transplantation, studies document that adult height is impaired in about 40% of children with stage 5 chronic kidney disease who had undergone kidney transplantation. The use of rhGH has been shown to improve linear growth in childhood chronic kidney disease stage 3–5, in conjunction with management of nutritional issues and metabolic abnormalities.
Due to concerns about the safety profile, in particular, the risk of transplant rejection, rhGH is generally discontinued following kidney transplant. The role of treatment with rhGH prior to kidney transplant is still poorly understood. The authors in this research performed a prospective observational study aiming to evaluate linear growth following kidney transplant, in particular, studying the role of treatment with rhGH before kidney transplant. From a total of 947 children who received a kidney transplant from May 1998 to January 2020, a total of 146 prepubertal children were included in this study – 52 children received rhGH prior to kidney transplant, and 94 did not receive rhGH prior to kidney transplant. Of the 94 children who did not receive rhGH before kidney transplant 17 were treated with rhGH post transplant. Following 7 years after kidney transplant, the height z-score was significantly higher in the group that received rhGH pretransplant (-0.85 vs. -1.76). The group that did not receive rhGH pretransplant was noted to have a faster decline in transplant function, lower hemoglobin, higher C-reactive protein, and higher steroid exposure. While the results of this study seem to demonstrate that rhGH therapy before transplant in children with chronic kidney disease is associated with improvement in height, further studies are needed to evaluate if this improvement in height is also related to disease-related factors. The role of rhGH in kidney-related factors, inflammation, anemia, and nutritional factors should also be investigated in future studies

Effects of vitamin D and high dairy protein intake on bone mineralization and linear growth in 6- to 8-year-old children: the D-pro randomized trial

Comments: It is known that vitamin D and dairy protein may improve bone mass accrual during childhood but also have a role in improving linear growth. The global increase in vitamin D deficiency is well documented. Existing recommendations in Nordic countries and the USA suggest that supplementation with vitamin D of 10 µg/day and 15 µg/day is needed. However, a recent meta-analysis suggests that 20 µg/day may be needed to maintain 25 hydroxy-vitamin D levels of greater than 50 nmol/L. Other than vitamin D supplementation, milk and dairy proteins could also improve linear growth and bone mineralization.
This clinical trial aimed to evaluate the combined and separate effects of vitamin D supplementation and high-protein or normal-protein yogurt on linear growth and bone health during the winter months in 6- to 8-year-old healthy children. The primary outcome was DXA total body less head bone mineral density. Secondary outcome measures evaluated included other DXA parameters including at lumbar spine, height, and biomarkers of bone turnover and linear growth. Participants were randomized to 20 µg/day of vitamin D or placebo, and to substitute 260 g/day dairy with high-protein yogurt (10 g protein/100 g) or normal-protein yogurt (3.5 g/100 g). Vitamin D supplementation led to a greater increase in total body less head bone mineral content and lumbar spine bone mineral density. DXA bone parameters and bone biomarker of bone formation (osteocalcin) were lower in the high-protein groups compared with the normal-protein groups. In summary, this clinical trial showed that supplementation of 20 µg/day of vitamin D in healthy 6- to 8-year-olds improved bone mass in the whole body and lumbar spine and can be recommended. However, there is no evidence for the introduction of high protein on the clinical outcomes.

Dynamic changes in serum IGF-1 and growth during infancy: associations to body fat, target height and PAPPA2 genotype

Comments: IGF-1 is an important mediator of linear growth throughout childhood. In the infancy phase of growth, it is believed that IGF-1 may play a relatively smaller role in linear growth, although to date there are limited studies. In addition, normative data on IGF- 1 and IGF binding protein 3 (IGFBP-3) in infancy are lacking. Recently, cord blood concentrations of pregnancy plasma protein A2 (PAPPA2) were negatively associated with birth weight and birth length. Another recent study also identified an association between PAPPA2 and height in children. PAPPA2 cleaves IGF-1 from its binding proteins and thereby leads to a greater increase in bioactive IGF-1. 
The investigators in this study aim to evaluate the determinants of change in weight and length during infancy and to evaluate the impact of factors like IGF-1 and PAPPA2 on weight gain and length gain. A total of 233 healthy children (114 girls) were included in this study. All had repeated blood sampling throughout the first year of life. IGF-1 decreased during the first year of life in both boys and girls, whereas IGFBP-3 remained stable. Both IGF-1 and IGFBP-3 were associated with weight gain but not increase in length. This association was only noted in girls when the group was separated. The PAPPA2 genotype did not have any influence on weight gain or length. 
This study, therefore, suggests that the role of systemic factors like IGF-1 and IGFBP-3 is on an increase in mass or parameters of body composition rather than on an increase in bone length. Further studies should evaluate the effect of nutrition in the relationship between systemic factors like IGF-1 and IGFBP-3 and weight gain in infancy. This study also provided useful information on normative data of IGF-1 and IGFBP-3 in infancy.

The effects of nutrition on linear growth

Comments: This recent review summarizes information on systemic endocrine factors and nutritional factors that regulate linear growth during childhood. The role of key macronutrients and micronutrients is included, as are endocrine regulations like the GH/IGF-1 axis and insulin but also other factors like leptin and FGF21. A brief summary of clinically relevant states was included in the review, for example, anorexia nervosa and obesity. A multitude of chronic childhood conditions are also implicated and are areas of focus for clinical researchers in this field.

References

1. Lebenthal Y, Yackobovitch-Gavan M, Lazar L, Shalitin, S, Tenenbaum A, Shamir R, et al. Effect of a nutritional supplement on growth in short and lean prepubertal children: a prospective, randomized, double-blind, placebo-controlled study. J Pediatr. 2014;165:1190–93.e1.
2. Yackobovitch-Gavan M, Lebenthal Y, Lazar L, Shalitin, S, Demol S, Tenenbaum A, et al. Effect of nutritional supplementation on growth in short and lean prepubertal children after 1 year of intervention. J Pediatr. 2016;179:154–9.e1.
3. Wei J, Shimazu J, Makinistoglu MP, Maurizi A, Kajimura D, Zong H, et al. Glucose uptake and Runx2 synergize to orchestrate osteoblast differentiation and bone formation. Cell. 2015;161:1576–91.
4. Lee SY, Abel ED, Long F. Glucose metabolism induced by Bmp signaling is essential for murine skeletal development. Nat Commun. 2018;9:4831.
5. Hardeland R. Melatonin and the programming of stem cells. Int J Mol Sci. 2022;23:1971.