Bone Collagen Research Articles

Effects of ultrasound on the structural and functional properties of sheep bone collagen

The study evaluated the effect of an ultrasound-assisted treatment on the structural and functional properties of sheep bone collagen (SBC). The type and distribution of SBC were analyzed by proteome (shotgun) technology combined with liquid chromatography-tandem mass spectrometry. Compared with pepsin extraction, the ultrasound-assisted treatment significantly increased the collagen extraction rate by 17.4 pp (P < 0.05). The characteristic functional groups and structural integrity of collagen extracted by both methods were determined via Fourier transform infrared spectroscopy, ultraviolet absorption spectroscopy, and fluorescence spectroscopy. Circular dichroism spectra revealed that the ultrasound-assisted pretreatment reduced α-helix content by 1.6 pp, β-sheet content by 21.9 pp, and random coils content by 28.4 pp, whereas it increased β-turn content by 51.9 pp (P < 0.05), compared with pepsin extraction. Moreover, ultrasound-assisted treatment collagen had superior functional properties (e.g., solubility, water absorption, and oil absorption capacity) and foaming and emulsion properties, compared with pepsin extraction. Furthermore, the relative content of type I collagen in ultrasound-assisted extracted SBC was highest at 79.66%; only small proportions of type II, VI, X, and XI collagen were present. Peptide activity analysis showed that SBC had potential antioxidant activity, dipeptidyl peptidase 4 inhibitory activity, and angiotensin-converting enzyme inhibitory activity; it also had anticancer, antihypertensive, anti-inflammatory, and immunomodulatory effects.

Characterising the cave bear Ursus spelaeus Rosenmüller by ZooMS: a review of peptide mass fingerprinting markers

ABSTRACTIn the last decade, the identification of bone fragments by peptide mass fingerprinting or zooarchaeology by mass spectrometry is developing as a powerful tool in Quaternary palaeontology. The sequence of amino acids that make up the bone collagen molecule shows slight variations between taxa, which can be studied by mass spectrometry for taxonomic purposes. This requires reference databases that allow peptide identification. Although the cave bear (Ursus spelaeus Rosenmüller, 1794) is a common component in many European Pleistocene cave sites, no peptide fingerprint taxonomic study has paid special attention to this species up to now. For peptide markers in Ursidae, the most recent proposal is based on collagen obtained from a modern brown bear sample. In this work we attempt to cover this gap by studying bone collagen of cave and brown bear samples from different origins and different chronology, applying matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF). We also performed an in-silico study of ursid bone collagen sequences published in databases. In our results we detected some discrepancies between the peptides obtained from both in silico and MALDI TOF analysis of fossil collagen and those published in the literature, in which we conclude that there are some misidentified peptides. The identification of skeletal remains by means of their peptide fingerprint is proving to be a powerful tool in palaeontology, which will bear greater fruit once the limitations of a technique that is in its initial stages have been overcome.

The Bioaccessibility of Yak Bone Collagen Hydrolysates: Focus on Analyzing the Variation Regular of Peptides and Free Amino Acids

The lack of a bioaccessibility test for yak bone collagen hydrolysates (YBCH) limits their development as functional foods. In this study, simulated gastrointestinal digestion (SD) and absorption (SA) models were utilized to evaluate the bioaccessibility of YBCH for the first time. The variation in peptides and free amino acids was primarily characterized. There was no significant alteration in the concentration of peptides during the SD. The transport rate of peptides through the Caco-2 cell monolayers was 22.14 ± 1.58%. Finally, a total of 440 peptides were identified, more than 75% of them with lengths ranging from 7 to 15. The peptide identification indicated that about 77% of the peptides in the beginning sample still existed after the SD, and about 76% of the peptides in the digested YBCH could be observed after the SA. These results suggested that most peptides in the YBCH resist gastrointestinal digestion and absorption. After the in silico prediction, seven typical bioavailable bioactive peptides were screened out and they exhibited multi-type bioactivities in vitro. This is the first study to characterize the changes in peptides and amino acids in the YBCH during gastrointestinal digestion and absorption, and provides a foundation for analyzing the mechanism of YBCH's bioactivities.

Berberine derivative WJCPR11 enhances osteoblast differentiation

Berberine is a phytocompound from plants viz. Phellodendri cortex and Coptis rhizome, used to treat a variety of diseases. It is effective in preventing osteoporosis, but it is less effective than drugs currently used in clinical practice. In this study, we used a novel berberine derivative, WJCPR11, to promote osteoblast differentiation and to investigate its use in the prevention and treatment of osteoporosis. WJCPR11 at a safe concentration without toxicity increased alkaline phosphatase (ALP) activity induced by bone morphogenetic protein 2 (BMP2) dose-dependently. The mRNA expression of ALP, osteocalcin (OC), runt-related transcription factor 2 (Runx2), and osterix was increased, with the ALP level increasing the most. In addition, the protein abundance of bone sialoprotein (BSP), collagen, type I, alpha 1, Runx2, and osterix were also increased. Moreover, the transcriptional activity of ALP, BSP, and OC was increased by WJCPR11, with OC showing the most significant increase. The results indicate that osteoblast differentiation is promoted by WJCPR11, and it could play a role in the prevention of osteoporosis.

Biomimetic Remineralized Three-Dimensional Collagen Bone Matrices with an Enhanced Osteostimulating Effect.

Bone grafts with a high potential for osseointegration, capable of providing a complete and effective regeneration of bone tissue, remain an urgent and unresolved issue. The presented work proposes an approach to develop composite biomimetic bone material for reconstructive surgery by deposition (remineralization) on the surface of high-purity, demineralized bone collagen matrix calcium phosphate layers. Histological and elemental analysis have shown reproduction of the bone tissue matrix architectonics, and a high-purity degree of the obtained collagen scaffolds; the cell culture and confocal microscopy have demonstrated a high biocompatibility of the materials obtained. Adsorption spectroscopy, scanning electron microscopy, microcomputed tomography (microCT) and infrared spectroscopy, and X-ray diffraction have proven the efficiency of the deposition of calcium phosphates on the surface of bone collagen scaffolds. Cell culture and confocal microscopy methods have shown high biocompatibility of both demineralized and remineralized bone matrices. In the model of heterotopic implantation in rats, at the term of seven weeks, an intensive intratrabecular infiltration of calcium phosphate precipitates, and a pronounced synthetic activity of osteoblast remodeling and rebuilding implanted materials, were revealed in remineralized bone collagen matrices in contrast to demineralized ones. Thus, remineralization of highly purified demineralized bone matrices significantly enhanced their osteostimulating ability. The data obtained are of interest for the creation of new highly effective osteoplastic materials for bone tissue regeneration and augmentation.

Stable isotope analyses of amino acids reveal the importance of aquatic resources to Mediterranean coastal hunter-gatherers.

Determining the degree to which humans relied on coastal resources in the past is key for understanding long-term social and economic development, as well as for assessing human health and anthropogenic impacts on the environment. Prehistoric hunter-gatherers are often assumed to have heavily exploited aquatic resources, especially those living in regions of high marine productivity. For the Mediterranean, this view has been challenged, partly by the application of stable isotope analysis of skeletal remains which has shown more varied coastal hunter-gatherer diets than in other regions, perhaps due to its lower productivity. By undertaking a more specific analysis of amino acids from bone collagen of 11 individuals from one of the oldest and best-known Mesolithic cemeteries in the Mediterranean, at El Collado, Valencia, we show that high levels of aquatic protein consumption were achieved. By measuring both carbon and nitrogen in amino acids, we conclude that some of the El Collado humans relied heavily on local lagoonal fish and possibly shellfish, rather than open marine species. By contrast to previous suggestions, this study demonstrates that the north-western coast of the Mediterranean basin could support maritime-oriented economies during the Early Holocene.

Functional Properties of Collagen Extracted From Catfish ( Silurus triostegus) Bone

Back ground: Collagen accounts for 25percent of all proteins found in vertebrates. The words &amp;quot;collagen&amp;quot;is created with Greek words &amp;quot;kolla&amp;quot; and &amp;quot;genos,&amp;quot; which respectively mean &amp;quot;glue&amp;quot; and &amp;quot;formation.&amp;quot; It hasa wide range of biological and pharmacological uses. Pig and cow skins provide the majority of thecollagen. Utilizing animal collagen has been restricted as a result of epidemics of certain animal diseasesincluding foot-and-mouth disease (FMD) and bovine spongiform encephalopathy (BSE) (FMD), whichpose a risk of transmission to humansObjective: The goal of this study is to extract collagen from catfish bone.Material and methods: Catfish (Silurus triostegus) bone was used to extract collagen that has beenpepsinized and acid-solubilized collagen (ASC) (PSC). The extracts&amp;#39; proximate composition (moisture,protein, fat, and ash) was measured, and dry weight was used to compute the yield of ASC and PSC. Themolecular weight of experimental collagen ranged from 97 to 200 KD, according to the SDS-PAGEpattern. In order to identify bone collagen, the Fourier Transform Infrared (FTIR) spectra analysis for theacquired ASC and PSC, as well as the standard collagen (type I, II), was used. Using a scanning electronmicroscope (SEM, the morphological structure of ASC and PSC was revealed), and the diffraction angles(2 θ) of ASC and PSC were determined using X-ray analysis. For quality control, the HPLC techniquewas used to determine the collagen.Rustles: The proximate composition of ASC and PSC bone of catfish. The ASC bone of catfish containedmoisture (75.22%), good amount of protein (16.65%) , fat (2.40%) and ash content (0.28 %), While thePSC bone had slightly lower moisture (68.50%) than ASC, it had higher protein (19.93%), fat (1.825%),and ash (0.58%) levels ,HPLC SEM ,FTIR ,Electrophoresis, X-ray.Conclusion: Fish bone was promising sources for preparing collagen, acid soluble collagen (ASC),pepsin soluble collagen (PSC) were successfully extracted by two methods. The collagen extracted frombone catfish, was of type I collagen, which are composed two α1, one α2 chains, and β chains as shownby SDS-PAGE patterns.

Biochemical and Microstructural Characteristics of Collagen Biopolymer from Unicornfish (Naso reticulatus Randall, 2001) Bone Prepared with Various Acid Types

Biopolymer-like collagen has great industrial potential in terms of its excellent properties, such as strong biocompatibility, high degradability, and low antigenicity. Collagen derived from fish by-products is preferable as it is safer (free from transmittable diseases) and acceptable to most religious beliefs. This study aimed to characterize the unicornfish (Naso reticulatus Randall, 2001) bone collagens prepared with different type of acids, i.e., acetic acid, lactic acid, and citric acid. A higher yield (Y) (p < 0.05) was obtained in the citric-acid-soluble collagen (CASC) (Y = 1.36%), followed by the lactic-acid-soluble collagen (LASC) (Y = 1.08%) and acetic-acid-soluble collagen (AASC) (Y = 0.40%). All extracted collagens were classified as type I due to the presence of 2-alpha chains (α1 and α2). Their prominent absorption spectra were located at the wavelengths of 229.83 nm to 231.17 nm. This is similar to wavelengths reported for other fish collagens. The X-ray diffraction (XRD) and infrared (IR) data demonstrated that the triple-helical structure of type I collagens was still preserved after the acid-extraction process. In terms of thermal stability, all samples had similar maximum transition temperatures (Tmax = 33.34-33.51 °C). A higher relative solubility (RS) of the unicornfish bone collagens was observed at low salt concentration (0-10 g/L) (RS > 80%) and at acidic condition (pH 1.0 to pH 3.0) (RS > 75%). The extracted collagen samples had an irregular and dense flake structure with random coiled filaments. Overall, bones of unicornfish may be used as a substitute source of collagen.

Investigation of consumption practices in the early Mycenaean period through biomolecular analyses: The case of the Ayios Vasileios North Cemetery (Laconia)

The Early Mycenaean cemetery of Ayios Vasileios in Laconia offers unique insights into social practices at the dawn of the Late Bronze Age (ca. 1700 1500BCE). The North Cemetery consists mostly of large and carefully constructed cist tombs, but the majority of burials were unfurnished. Although a number of studies are engaged in the investigation of this formative period, integrated approaches which focus on the social practice of consumption and contextualised understanding of material culture are still scarce. In this paper we present the results of an integrated study of ceramic vessel use, implementing gas chromatography - mass spectrometry (GC–MS) and a detailed typological characterisation of the pottery recovered, as well as correlations drawn with the results from isotope analyses on the skeletons. Our aim is to explore the ways technological, ecological, and social factors affect consumption in the mortuary sphere. Ceramic offerings are few in number and consist only of one or two small vases per burial, usually a drinking cup and a small jar, but variety in style and fabric suggests increased interaction with areas further afield. Organic residue analysis shows that several of the vessels in the graves had been used before deposition and contained lipids from animal sources and plant lipids. Isotope analyses on bone collagen indicate the exploitation of similar types of food sources by the living community, with little observable variation.New perspectives are offered for better understanding of the social practices in Early Mycenaean Laconia, while reflecting on everyday life and subsistence by drawing together multiple lines of analysis.

New radiocarbon dates and the prehistoric human occupation at the Lake Qinghai Basin, northeast Tibetan Plateau

The Tibetan Plateau is known as the rooftop of the planet. Its hypoxic atmosphere in low oxygen partial pressure, extreme seasonal climate variation, and aridity poses severe challenges as harsh living environments for prehistoric human to occupy in such high-altitude niches. However, the Lake Qinghai is the largest lake on the Tibetan Plateau and the basin has rich water, mineral, and wild food resources, and prehistoric human occupied the Lake Qinghai Basin long before local farming was in practice. In this paper we present results of faunal assemblages associated with variable stone artifacts and pottery shards with bone collagen accelerator mass spectrometry radiocarbon (AMS 14C) dates from 3 new archaeological sites to investigate human occupation history in the southern Lake Qinghai Basin. The AMS 14C dating results reveal that the occupation occurs intermittently from the late Glacial to entire Holocene. Through compilation and synthesis of regional archaeological records, we found that prehistoric humans went through 3 phases of subsistence strategies: 1) hunting large and medium ungulates; 2) hunting medium ungulates; 3) foraging small size mammals with birds and fish diets. At the end, prehistoric humans increasingly adapted to forage and consume caprinaes species (goat), which represents a precursor of domestication or semi-domestication of wild animals in the Lake Qinghai Basin. We further attribute the utilization of caprinaes animal resources to the lower limbs of toe and phalange bones with low nutritious values to advanced social organization that implied prehistoric humans separated hunting activity from protein processing practices. Our investigation of zooarchaeology with reliable bone collagen AMS 14C dates could become a novel approach to study adaptation strategy and social structure changes of prehistorical Tibetan humans.

Proline promotes proliferation and drug resistance of multiple myeloma by downregulation of proline dehydrogenase.

Amino acids in the bone marrow microenvironment (BMME) are a critical factor for multiple myeloma (MM) progression. Here, we have determined that proline is elevated in BMME of MM patients and links to poor prognosis in MM. Moreover, exogenous proline regulates MM cell proliferation and drug resistance. Elevated proline in BMME is due to bone collagen degradation and abnormal expression of the key enzyme of proline catabolism, proline dehydrogenase (PRODH). PRODH is downregulated in MM patients, mainly as a result of promoter hypermethylation with high expression of DNMT3b. Thus, overexpression of PRODH suppresses cell proliferation and drug resistance of MM and exhibits therapeutic potential for treatment of MM. Altogether, we identify proline as a key metabolic regulator of MM, unveil PRODH governing MM progression and provide a promising therapeutic strategy for MM treatment.

SAMPLE PREPARATION METHODS USED AT THE AUSTRALIAN NATIONAL UNIVERSITY RADIOCARBON FACILITY

ABSTRACTThe Single Stage AMS radiocarbon facility at the Australian National University has operated for the past 14 years. This paper presents the pretreatment methods used for the major sample types dated and reflects on whether quality assurance protocols can adequately detect altered materials. The majority of fossil samples dated by the facility are from tropical and arid environments where diagenesis of both organic samples and carbonates is often severe. A large proportion of the samples submitted cannot be dated, and screening and quality assurance methods are crucial. Based on analysis of 250 measurements on bone collagen, quality assurance indicators for bone are revised. From May 2021, the laboratory has accepted dates on collagen where yield &gt;0.5%, %C is in the range of 39.7–46.9, and the C:N ratio is between 3.00 and 3.30.

Antifatigue and microbiome reshaping effects of yak bone collagen peptides on Balb/c mice

As the main by-product of yak (Bos grunniens) processing, yak bone was discarded or simply processed for low-value production. The development of yak bone collagen peptides (YBP) has shown great potential. In the study, the 1 g/kg· BW peptides were supplied (the group of YBP) or not supplie the control group) in the Balb/c mice diet for 18 weeks. Peptide supplementation prolonged the swimming time of mice by 25% and 37% for twice tests, respectively, accompanied by the increased content of glucose (Glu), lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione (GSH), and decreased content of lactic acid (LA), blood ammonia (BA) in blood. YBP intervention promoted the relative abundance of Bacteroides, Blautia, Muribaculum, etc. The proportion of amino acid and carbohydrate transport and metabolism increased among all the predicted functions from the orthologous groups of proteins (COG) database. Besides that, after YBP intervention, many up-regulated proteins in the liver were involved in the protein and carbohydrate metabolism-related pathways, and the proteins in lipid metabolism-related pathways were down-regulated. This study firstly clarified the antifatigue and gut microbiome reshaping effects of YBP on Balb/c mice and provided the theoretical basis for future development.

Evaluating the efficacy of collagen isolation using stable isotope analysis and infrared spectroscopy

Stable isotope analysis of bone and dental collagen is one of the most common methods to investigate the ecology of modern and extinct human and animal populations. However, since bone and dentine are composite materials with both organic and mineral components, the mineral component must be removed prior to analysis. In this study we investigated the timing and efficacy of mineral removal from bone and dentine. We performed a series of time-step experiments that show that mineral removal can be quantified over short periods of time using Fourier Transform Infrared Spectroscopy (FTIR), and collagen alteration can be tracked using a combination of stable isotope analysis and elemental analysis. We tested our methods on three modern materials: mammalian bone, mammalian dentine, and shark dentine. Our results show: 1) mineral removal is a necessary step, as structural carbonate has a strong influence on stable isotope compositions; 2) demineralization using weak acid (0.1 M HCL) does not appear to alter the elemental and isotopic compositions of collagen. Our methods can be used as a framework to evaluate the need-for and efficacy of other demineralization methods in use today including EDTA-demineralization and lipid removal.

High-resolution Bayesian chronology of the earliest evidence of domesticated animals in the Dutch wetlands (Hardinxveld-Giessendam archaeological sites).

The archaeological sites of Hardinxveld-Giessendam de Bruin and Polderweg, situated in the Rhine-Meuse delta, are the best-preserved Mesolithic sites in the Netherlands. Due to the early appearance of domesticated animals in their faunal assemblage, they are also integral to the research of the emergence of animal husbandry in the region. This study focuses on the precise chronology of the sites, using radiocarbon dating and Bayesian modelling of both newly acquired and legacy radiocarbon dates. To mitigate the risk of erroneous dates, we dated the bone collagen of 26 herbivorous and one aquatic mammals from clear archaeological contexts and discovered that the most recent occupational phases at both sites are several centuries younger than previously thought. This is consistent with material evidence of lifestyle changes in the final phase at Hardinxveld-Giessendam de Bruin, which is now, according to our chronology, contemporaneous with the similar patterns produced in the region.

Yak bone collagen-derived anti-inflammatory bioactive peptides alleviate lipopolysaccharide-induced inflammatory by inhibiting the NF-κB signaling pathway and nitric oxide production

Anti-inflammatory peptides that derived from food attract more and more attention due to their wide range of sources and easy absorption. In this study, the aim was to identify and characterize anti-inflammatory peptides from yak bone collagen. The prepared yak bone collagen peptides (YBCPs) was separated, purified and identified through ultrafiltration, reverse-phase high-performance liquid chromatography (RP-HPLC), and nano-liquid chromatography–tandem mass spectrometry (nano LC-MS/MS) successively. Finally, a total of 115 peptides were identified. After that, 12 peptides were screened out based on the predicted biological activity score. Molecular docking results indicated that 6 peptides (GPAGPSGPAGK, GPAGPSGPAGKDGR, GPSGPQGIR, GPAGPQGPR, GEAGPAGPAGPAGPR, and GEGGPQGPR) could successfully interact with the key factors in the nuclear factor kappa-B (NF-κB) signaling pathway and nitric oxide (NO) production due to form various bonds such as salt bridge, conventional hydrogen bond, and carbon-hydrogen bond. The anti-inflammatory effects of these 6 peptides were further verified by in vitro cell test. Results indicated that the 6 peptides might play an anti-inflammatory role by regulating different proteins in the NF-κB signaling pathway and NO production. This study would further promote the application of yak bone collagen in health food.

Variability in feeding habitats of red deer sensu lato in Eurasia in the Late Pleistocene and Holocene

Red deer (Cervus elaphus) is one of the species that is rather wide spread and survived across Europe over the Holocene. The analyses of carbon and nitrogen stable isotopes in bone collagen of ungulate remains have been applied in paleoecological studies as environmental and dietary indicators. In this study we present the carbon and nitrogen stable isotope compositions of previously radiocarbon-dated red deer bone samples (N = 68) found in Central, Southern and Eastern Europe and Asia and aligned to one of two species: European red deer (Cervus elaphus) and wapiti (Cervus canadensis). We showed that the values of carbon and nitrogen stable isotope ratios of European red deer and wapiti overlapped. Among all analysed independent factors (determined for the locality and time period relevant for each of the analysed samples), the variability of δ13C values in European red deer dated to the Holocene is best explained by forest cover and mean July temperature, and variability of δ15N values by the mean July temperature, annual precipitation and altitude. Additionally, combining the results of the present study with isotopic data on European red deer collected from published sources, we revealed that the values of δ13C and of δ15N in C. elaphus bones changed according to environmental oscillations that took place in Europe over the last 50 000 years. We concluded that red deer shifted their feeding habitats in relation to changing environmental conditions, for example, forest expansion during the climate warming, and in the mid to later Holocene in response to deforestation caused by human activity and the spread of agriculture. We also found out that red deer reacted in varied ways to changing local conditions in different regions of Europe. Modern individuals of C. elaphus had the lowest δ13C values among all analysed specimens, so they probably inhabited the most densely forested areas in comparison to other European red deer populations during the last 50 000 years.

Advances in osseointegration of biomimetic mineralized collagen and inorganic metal elements of natural bone for bone repair.

At this stage, bone defects caused by trauma, infection, tumor, or congenital diseases are generally filled with autologous bone or allogeneic bone transplantation, but this treatment method has limited sources, potential disease transmission and other problems. Ideal bone-graft materials remain continuously explored, and bone defect reconstruction remains a significant challenge. Mineralized collagen prepared by bionic mineralization combining organic polymer collagen with inorganic mineral calcium phosphate can effectively imitate the composition and hierarchical structure of natural bone and has good application value in bone repair materials. Magnesium, strontium, zinc and other inorganic components not only can activate relevant signaling pathways to induce differentiation of osteogenic precursor cells but also stimulate other core biological processes of bone tissue growth and play an important role in natural bone growth, and bone repair and reconstruction. This study reviewed the advances in hydroxyapatite/collagen composite scaffolds and osseointegration with natural bone inorganic components, such as magnesium, strontium and zinc.

Combining Bone Collagen Material with hUC-MSCs for Applicationto Spina Bifida in a Rabbit Model.

Spina bifida is one of the neural tube defects, with a high incidence in human birth defects, which seriously affects the health and quality of life of patients. In the treatment of bone defects, the source of autologous bone is limited and will cause secondary damage to the patient. At the same time, since the bone tissue in animals needs to play a variety of biological functions, its complex structure cannot be replaced by a single material. The combination of mechanical materials and biological materials has become a common choice. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have the advantages of easy access, rapid proliferation, low immunogenicity, and no ethical issues. It is often used in the clinical research of tissue regeneration and repair. Therefore, in this study, we established a spina bifida model using Japanese white rabbits. This model was used to screen the best regenerative repair products for congenital spina bifida, and to evaluate the safety of regenerative repair products. The results showed that the combination of hUC-MSCs with collagen material had better regeneration effect than collagen material alone, and had no negative impact on the health of animals. This study provides a new idea for the clinical treatment of spina bifida, and also helps to speed up the research progress of regenerative repair products.

Children of the grave: Investigating non-adult feeding practices in medieval and early modern Estonia through stable isotope analysis.

Studying infant diet and feeding practices through stable isotope analysis provides direct insight into the life and health of vulnerable population groups in the past. Although the general diet in medieval and early modern Livonia has been reconstructed from written sources, little is known about childhood diet during this tumultuous period of Eastern European history. This study presents a comparative investigation of the staple non-adult diet in urban/rural communities during the 13th-17th centuries AD, with a special focus on feeding practices. We aim to reveal the impact of socio-economic circumstances on early childhood nutrition, which affects the physical development and overall survival of this susceptible population group. Bone collagen samples from 176 individuals between the fetal and the 7-15 age categories from four urban/rural South-Estonian cemeteries were cross-sectionally analyzed via EA-IRMS (Elemental Analysis with Isotope Ratio Mass Spectroscopy) for δ13C and δ15N. Results suggest that South-Estonian children had a staple terrestrial C3 diet integrated with animal proteins. Significant divergences were observed between urban and rural sites and slight variation occurred among rural subgroups, possibly resulting from a wider food choice available in towns, different consumption of C4 foods, and/or secular changes. This study provides the first data regarding infant feeding practices in medieval and early modern Livonia. These practices were similar among the different contexts, indicating comparable cultural traditions in child rearing. Breastfeeding was likely practiced for 1-2 years, with supplementary foods introduced around 1 year of age. The weaning process was probably concluded around the age of 3. The δ13C and δ15N values of older children are comparable to those of the adults from the same sites, indicating their diets became similar after weaning, when they started working and obtained a more mature social status.