Collagen-Derived Dipeptide Pro-Hyp Enhanced ATDC5 Chondrocyte Differentiation under Hypoxic Conditions.

Chondrocytes are surrounded by a lower oxygen environment than other well-vascularized tissues with higher oxygenation levels. Prolyl-hydroxyproline (Pro-Hyp), one of the final collagen-derived peptides, has been previously reported to be involved in the early stages of chondrocyte differentiation. However, whether Pro-Hyp can alter chondrocyte differentiation under physiological hypoxic conditions is still unclear. This study aimed to investigate whether Pro-Hyp affects the differentiation of ATDC5 chondrogenic cells under hypoxic conditions. The addition of Pro-Hyp resulted in an approximately 18-fold increase in the glycosaminoglycan staining area compared to the control group under hypoxic conditions. Moreover, Pro-Hyp treatment significantly upregulated the expression of SOX9, Col2a1, Aggrecan, and MMP13 in chondrocytes cultured under hypoxic conditions. These results demonstrate that Pro-Hyp strongly promotes the early differentiation of chondrocytes under physiological hypoxic conditions. Therefore, Pro-Hyp, a bioactive peptide produced during collagen metabolism, may function as a remodeling factor or extracellular matrix remodeling signal that regulates chondrocyte differentiation in hypoxic cartilage.

Body knowledge in children with spina bifida.

BACKGROUND: Patients with spina bifida suffer from motor paralysis and sensory disturbance, secondary deformation of the lower extremities, and development of decubitus ulcers. A deep understanding of one's body, such as identifying the names, functions, relationships, homology (e.g. upper and lower limbs) and relative position of body parts, may prevent secondary disorders. The awareness of the body and its characteristics in children with spina bifida has not been fully elucidated. This study aimed to investigate how children with spina bifida perceive their bodies, especially their paralyzed parts and related or homologous ones. METHODS: Participants included 36 children with spina bifida and 14 control children, aged 5-11 years. They were asked to draw self-portraits, and to answer questions about the names of body parts. The number of drawn body parts in the pictures and the correct response rates to the questions were quantified and compared between children with spina bifida and control children. RESULTS: Children with spina bifida drew fewer hands, legs, and feet than control children. In the verbal tests, children with spina bifida had a lower correct response rate than control children on questions concerning the upper limbs, hands, trunk, and feet. CONCLUSIONS: Children with spina bifida have diminished visuospatial and lexical-semantic body knowledge of the paralyzed parts and related organs.

Visual attention to their own paralytic limbs in children with spina bifida: Measurement of gaze direction using eye tracking.

BACKGROUND: Patients with spina bifida experience sensory and motor paralysis and complications in the form of deformation and skin problems of the lower limbs. Enhancing their awareness of the paralysed lesions could be helpful for these patients to prevent secondary disorders. This study sought to investigate to what extent children with spina bifida are visually aware of their body parts and, in particular, to their paralysed lesions. METHODS: Participants included children with spina bifida (n = 10) and typically developing control children (n = 10) between the ages of 5 and 11 years. All participants were shown still images of themselves on a monitor after which their gaze direction was measured using an eye tracker, with or without providing an instruction to direct their attention to their limbs. The gaze direction data were analyzed and compared between children with spina bifida and the control children. RESULTS: Children with spina bifida paid visual attention to their bodies in a manner similar to that of control children. Prompts to direct their attention to their limbs were effective in both groups; however, the degree of efficacy in children with spina bifida was inferior to that in control children. CONCLUSIONS: Promotion of visual awareness to their paralytic parts could be a reasonable and effective method for children with spina bifida to improve their understanding and recognition of their body and prevent associated skin problems.

Treatment approaches for congenital transverse limb deficiency: Data analysis from an epidemiological national survey in Japan.

BACKGROUND: Congenital limb deficiency is a rare anomaly that impairs limb function. Transverse deficiency accounts for approximately half of congenital limb deficiency cases. In Japan, there have been no detailed data of clinical features, especially treatment approaches, of this disorder. The present study aimed to investigate the status of treatment approaches of congenital transverse limb deficiency in Japan. METHODS: From the national epidemiological survey of congenital limb deficiency undertaken in Japan in 2016, all the data of 200 patients with congenital transverse limb deficiencies were extracted. These data were analysed to reveal the treatment approaches of congenital transverse limb deficiency and its basic clinical features. RESULTS: Surgical treatments and prosthetic or orthotic intervention had been implemented or planned for about one-quarter of patients, respectively. In the upper limb deficiencies, prosthetic or orthotic intervention was likely chosen in cases of deficiency at the metacarpal or proximal to metacarpal level. Surgical treatment was chosen only in cases of deficiency at the carpal or distal to carpal level. Although the number of patients with transverse lower limb deficiencies was small, prosthetic or orthotic intervention was likely chosen in proximal deficiencies, and surgical treatment was likely chosen in distal deficiencies. CONCLUSIONS: Herein, we revealed the status of treatment approaches for congenital transverse limb deficiency in Japan. Approximately half of the patients had no history of-and no plans for-surgical, prosthetic, or orthotic interventions. Further treatment advances may enable patients with congenital limb deficiencies to increase their participation in daily activities. STUDY DESIGN: Cross-sectional survey.

Effect of prostheses on children with congenital upper limb deficiencies.

BACKGROUND: Individual weaknesses in motor skills are a characteristic of children with congenital upper limb deficiencies. These weaknesses increase with age. In Japan, however, prosthetic prescription and subsequent rehabilitation approaches for children with upper limb deficiencies are insufficient and often delayed. This study aimed to elucidate whether rehabilitation approaches, including prostheses prescription and occupational therapy, improve these children's adaptive behaviors, especially their motor skills. METHODS: The study included nine children, aged 0-6 years, with unilateral transradial or transcarpal upper limb deficiencies. We measured their adaptive behaviors and motor skills at the beginning of prosthetic therapy and after 1.5 years, using the Vineland Adaptive Behavior Scales - Second Edition. RESULTS: The score for the motor skills domain was significantly lower than the median score of the domains at the beginning of prosthetic therapy. The motor skill weaknesses significantly improved after 1.5 years of prosthetic therapy. CONCLUSIONS: Although children with congenital upper limb deficiencies have individual weaknesses in their motor skill behavior, it was shown that these weaknesses can be improved through rehabilitation approaches, including occupational and prosthetic therapies. Issuing the appropriate prostheses and implementing the appropriate training to use the prostheses for congenital upper limb deficiencies are reasonable and meaningful interventions to improve quality of life.

Collagen-derived dipeptide prolyl hydroxyproline directly binds to Foxg1 to change its conformation and inhibit the interaction with Runx2.

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression. Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp.

Body knowledge in children with congenital lower limb deficiency.

BACKGROUND: In order to effectively improve motor function, a sound understanding of one's body - for example, relative spatial position, relationships, names and functions of body parts - is essential. The aim of this study was to explore how children with congenital lower limb deficiency (LLD) perceive their bodies, particularly their legs. METHODS: Six children with congenital LLD and 14 controls, aged 5-12 years, were recruited for this study. They drew self-portraits and answered questions about names of body parts. These body part-related questions consisted of a production test, in which an examiner pointed to body parts on each child and asked the child to name them, and a comprehension test, in which the examiner mentioned body parts and asked the child to point to them on their own body. RESULTS: No differences were found between the self-portraits of children with LLD and those of the control children. In the verbal tests, children with LLD responded correctly at lower rates to questions on body trunk, upper limbs, arms, hands and feet than the control children. CONCLUSION: Children with LLD have diminished lexical-semantic body knowledge of the upper limbs and feet compared with children without LLD.

Congenital limb deficiency in Japan: a cross-sectional nationwide survey on its epidemiology.

BACKGROUND: Congenital limb deficiency is a rare and intractable disease, which impairs both function and appearance of the limbs. To establish adequate medical care, it is necessary to reveal the actual conditions and problems associated with this disease. However, there have been no extensive epidemiological surveys in Japan addressing this disease. This is the first nationwide epidemiological survey of congenital limb deficiency in this country. METHODS: With the cooperation of epidemiology experts, we performed a two-stage nationwide survey to estimate the number of patients with congenital limb deficiency and reveal basic patient features. We targeted orthopaedic surgery, paediatric, and plastic surgery departments. Hospitals were categorized according to the institution type and the number of hospital beds; hospitals were randomly selected from these categories. We selected 2283 departments from a total 7825 departments throughout Japan. In this study, we defined congenital limb deficiency as partial or total absence of the limbs, proximal to the proximal interphalangeal joint of the fingers/lesser toes or interphalangeal joint of the thumb/great toe. We distributed the first survey querying the number of initial patient visits from January 2014 to December 2015. Targets of the second survey were departments that reported one or more initial patient visits in the first survey. RESULTS: In the first survey, 1767 departments responded (response rate: 77.4%). Among them, 161 departments reported one or more initial patient visits. We conducted the second survey among these 161 departments, of which 96 departments responded (response rate: 59.6%). The estimated number of initial visits by patients with congenital limb deficiency was 417 (95% confidence interval: 339-495) per year in 2014 and 2015. The estimated prevalence of congenital limb deficiency in Japan was 4.15 (95% confidence interval: 3.37-4.93) per 10,000 live births. The sex ratio was 1.40. Upper limbs were more affected than lower limbs. CONCLUSIONS: We revealed the estimated number of initial patient visits per year and birth prevalence of congenital limb deficiency in Japan. Our results will contribute to establishing the disease concept and grades of severity of congenital limb deficiency.

Collagen-derived dipeptide prolyl-hydroxyproline promotes osteogenic differentiation through Foxg1.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. We previously reported that Pro-Hyp promotes the differentiation of osteoblasts by increasing Runx2, osterix and Col1α1 mRNA expression levels. Here, to elucidate the mechanism of Pro-Hyp promotion of osteoblast differentiation, we focus on the involvement of Foxo1 in osteoblast differentiation via Runx2 regulation and the role of Foxg1 in Foxo1 regulation. The addition of Pro-Hyp had no effect on MC3T3-E1 cell proliferation in Foxo1- or Foxg1-knockdown cells. In Foxo1-knockdown cells, the addition of Pro-Hyp increased ALP activity, but in Foxg1-knockdown cells, it had no effect on ALP activity. An enhancing effect of Pro-Hyp on the Runx2 and osterix expression levels was observed in Foxo1-knockdown cells. However, no enhancing effect of Pro-Hyp on osteoblastic gene expression was observed when Foxg1 was knocked down. These results demonstrate that Pro-Hyp promotes osteoblastic MC3T3-E1 cell differentiation and upregulation of osteogenic genes via Foxg1 expression.

Glucosamines Attenuate Bone Loss Due to Menopause by Regulating Osteoclast Function in Ovariectomized Mice.

The effect of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on bone metabolism in ovariectomized (OVX) mice was studied. After 12 weeks of feeding with 0.2% GlcN and 0.2% GlcNAc, the femoral bone mineral density in OVX mice was significantly increased compared with that in OVX mice fed the control diet. Histomorphometric analysis of the tibia indicated that the rates of osteogenesis and bone resorption were reduced due to the GlcN diet. The erosion depth of osteoclasts on the tibia in GlcN- and GlcNAc-fed OVX mice was significantly lower than that in the control OVX mice. The number of tartrate-resistant acid phosphatase-positive osteoclasts induced from bone marrow stem cells isolated from GlcN-fed OVX mice was significantly lower than that from control OVX mice. A loss of uterine weight and higher serum calcium concentration in the GlcN- and GlcNAc-fed OVX mice were observed. The results suggest that the intake of GlcN suppresses bone loss by inhibiting osteoclast differentiation and activity in a nonestrogenic manner.

Vitamin B12 deficiency-induced increase of osteoclastic bone resorption caused by abnormal renal resorption of inorganic phosphorus via Napi2a.

Vitamin B12 deficiency is a risk factor for bone disorders via mechanisms not fully understood. In this study, an increase in serum inorganic phosphorus (Pi) concentrations was associated with a vitamin B12 deficiency. Napi2a, a renal cotransporter for Pi reabsorption, accumulated on plasma membranes in a vitamin B12 deficiency suggests that vitamin B12 plays an important role in Pi homeostasis.

Oral collagen-derived dipeptides, prolyl-hydroxyproline and hydroxyprolyl-glycine, ameliorate skin barrier dysfunction and alter gene expression profiles in the skin.

Oral supplementation with collagen hydrolysate (CH) has been shown to improve the condition of the skin in humans and experimental animals. Several hydroxyproline-containing oligo-peptides were previously detected in human peripheral blood after the ingestion of CH, and the two dipeptides, prolyl-hydroxyproline (PO) and hydroxyprolyl-glycine (OG), have been proposed to have beneficial effects on human health. When HR-1 hairless mice were fed a HR-AD diet, which lacked magnesium and zinc, transepidermal water loss (TEWL) increased and water content of stratum corneum decreased. In the present study, we investigated the effects of dietary PO and OG on skin barrier dysfunction in HR-1 hairless mice. Mice were fed a HR-AD diet with or without PO (0.15%) and OG (0.15%) for 35 consecutive days. The administration of PO and OG significantly decreased TEWL, and significantly increased water content of stratum corneum. A DNA microarray analysis of the dorsal skin revealed differences in gene expression between the group administered PO and OG and the control group. We also identified muscle-related Gene Ontology as a result of analyzing the up-regulated genes. These results suggested that the administration of PO and OG improved skin barrier dysfunction and altered muscle-related gene expression.

Age-related decrease of IF5/BTG4 in oral and respiratory cavities in mice.

An IF5 cDNA was isolated by expression cloning from a mouse oocyte cDNA library. It encoded a protein of 250 amino acids, and the region of it encoding amino acids 1-137 showed 86.8% alignment with the anti-proliferative domain of BTG/TOB family genes. This gene is also termed BTG4 or PC3B. Transiently expressed IF5/BTG4 induced alkaline phosphatase activity in human embryonic kidney (HEK293T) and 2T3 cells. IF5/BTG4 mRNA was detected by reverse transcription polymerase chain reaction in pharynx, larynx, trachea, oviduct, ovary, caput epididymis, and testis, but not in lung, intestine, or liver. Immunohistochemistry showed the IF5/BTG4 protein to be present in epithelial cells of the tongue, palate, pharynx, internal nose, and trachea. Both protein and mRNA levels of IF5/BTG4 were reduced by aging when comparing 4-week-old mice with 48-week-old mice. Our findings suggest that IF5/BTG4 may be an aging-related gene in epithelial cells.

Neck range of motion measurements using a new three-dimensional motion analysis system: validity and repeatability.

PURPOSE: Neck movement is important for many activities of daily living (ADL). Neck disorders, such as cervical spondylosis and whiplash can limit neck movement and ADL. The cervical range of motion (CROM) device has been recently used to measure neck range of motion (ROM); however, this measurement includes trunk motion, and therefore does not represent a pure neck ROM measurement. The authors aimed to develop a new method to establish pure neck ROM measurements during flexion, extension, lateral bending, and rotation using a three-dimensional motion analysis system, VICON. METHODS: Twelve healthy participants were recruited and neck ROMs during flexion, extension, lateral bending, and rotation were measured using VICON and the CROM device. Test-retest repeatability was assessed using interclass correlation coefficients (ICCs), standard error of measurement (SEM), and minimal detectable change (MDC). Validity between two measurements was evaluated using a determination coefficient and Pearson's correlation coefficient. RESULTS: ICCs of neck ROM measured using VICON and the CROM device were all at substantial or almost perfect levels [VICON: ICC(1,2) = 0.786-0.962, the CROM device: ICC(1,2) = 0.736-0.950]. Both SEMs and MDCs were low in all measurement directions (VICON: SEM = 1.3°-4.5°, MDC = 3.6°-12.5°; the CROM device: SEM = 2.2°-3.9°, MDC = 6.1°-10.7°). Determination coefficients (R(2)s) and Pearson's correlation coefficients (rs) between the two measurement methods were high (R(2) = 0.607-0.745, r = 0.779-0.863). CONCLUSIONS: VICON is a useful system to measure neck ROMs and evaluate the efficacy of interventions, such as surgery or physiotherapeutic exercise.

[Bone and Nutrition. Functional foods for bone and cartilage tissues : Evidence for the action of collagen peptides].

Collagen peptides, including glucosamine, are well-known functional food materials that act on bone and cartilage tissues. Collagen peptides are used at a volume of about 6,000 tons per year as a raw material in Japan. Evidence for the action of collagen peptides for both bone and cartilage tissues has accumulated both at basic research level (in vivo and in vitro ) and clinically. To confirm the clinical effect of these peptides and clearly identify their mechanism of action at the molecular level, further clinical studies comprising disease-free subjects, alongside basic research studies, are required.

Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

Collagen-derived dipeptide prolyl-hydroxyproline cooperates with Foxg1 to activate the PGC-1α promoter and induce brown adipocyte-like phenotype in rosiglitazone-treated C3H10T1/2 cells.

Background: The global obesity epidemic is a significant public health issue, often leading to metabolic disorders such as diabetes and cardiovascular diseases. Collagen peptides (CP) and their bioactive component, Prolyl-hydroxyproline (Pro-Hyp), have shown potential in reducing adipocyte size, with unclear mechanisms concerning brown adipocyte differentiation. Methods: We investigated the effects of Pro-Hyp on the differentiation of brown adipocytes in C3H10T1/2 mesenchymal stem cells, focusing on its impact on adipocyte size, gene expression related to brown fat function, and mitochondrial activity. Results: Pro-Hyp treatment decreased adipocyte size and upregulated brown fat-specific genes, including C/EBPα, PGC-1α, and UCP-1. Remarkably, it did not alter PPARγ expression. Pro-Hyp also elevated mitochondrial activity, suggesting enhanced brown adipocyte functionality. A Pro-Hyp responsive element was identified in the PGC-1α gene promoter, which facilitated the binding of the Foxg1 transcription factor, indicating a novel regulatory mechanism. Conclusion: Pro-Hyp promotes brown adipocyte differentiation, potentially offering a therapeutic strategy for obesity management. This study provides a molecular basis for the anti-obesity effects of CP, although further in vivo studies are needed to confirm these findings and to investigate the potential impact on beige adipocyte differentiation.

Preventive Effects of Collagen-Derived Dipeptide Prolyl-Hydroxyproline against Dexamethasone-Induced Muscle Atrophy in Mouse C2C12 Skeletal Myotubes.

Glucocorticoids, commonly used to manage inflammatory diseases, can induce muscle atrophy by accelerating the breakdown of muscle proteins. This research delves into the influence of Prolyl-hydroxyproline (Pro-Hyp), a collagen-derived peptide, on muscle atrophy induced with dexamethasone (DEX), a synthetic glucocorticoid, in mouse C2C12 skeletal myotubes. Exposure to DEX (10 µM) for 6 days resulted in a decrease in myotube diameter, along with elevated mRNA and protein levels of two muscle-atrophy-related ubiquitin ligases, muscle atrophy F-box (MAFbx, also known as atrogin-1) and muscle ring finger 1 (MuRF-1). Remarkably, treatment with 0.1 mM of Pro-Hyp mitigated the reduction in myotube thickness caused by DEX, while promoting the phosphorylation of Akt, mammalian target of rapamycin (mTOR), and forkhead box O3a (Foxo3a). This led to the inhibition of the upregulation of the ubiquitin ligases atrogin-1 and MuRF-1. These findings indicate the potential significance of Pro-Hyp as a promising therapeutic target for countering DEX-induced muscle atrophy.

Relationship between degree of disability, usefulness of assistive devices, and daily use duration: an investigation in children with congenital upper limb deficiencies who use upper limb prostheses.

Upper limb prostheses can help children with congenital upper limb deficiencies (ULDs) perform activities of daily living. Although the degree of disability and prosthesis usefulness may be related to becoming accustomed to wearing a prosthesis, these relationships have not been confirmed. This study was aimed at investigating the relationship between motor function, the usefulness of prostheses in everyday activities, and daily duration of prosthesis use in children with congenital ULDs. Eleven children with congenital transcarpal or transradial ULDs and their caregivers participated in this study. The caregivers were asked to complete a questionnaire concerning their children's everyday activities and the daily duration of prosthesis wearing. The adaptive behavior scale was used to measure motor function prior to intervention. Correlations analyses revealed no significant correlation between the children's adaptive behavior, including motor skills before intervention, and daily duration of prosthesis wearing, but revealed a positive correlation between the number of activities that the children performed more effectively with their prostheses and the daily use duration. To ensure that children with congenital ULDs become accustomed to wearing their prostheses, the prostheses should meet the requirements of everyday activities.