A nutritional intervention study with hydrolyzed collagen in pre-pubertal Spanish children: influence on bone modeling biomarkers

Aim: The aim of the study was to investigate the influence of dietary intake of commercial hydrolyzed collagen (Gelatine Royal ®) on bone remodeling in pre-pubertal children. Methods: A randomized double-blind study was carried out in 60 children (9.42 ± 1.31 years) divided into three groups according to the amount of partially hydrolyzed collagen taken daily for 4 months: placebo (G-I, n = 18), collagen (G-II, n = 20) and collagen + calcium (G-III, n = 22) groups. Analyses of the following biochemical markers were carried out: total and bone alkaline phosphatase (tALP and bALP), osteocalcin, tartrate-resistant acid phosphatase (TRAP), type I collagen carboxy terminal telopeptide, lipids, calcium, 25-hydroxyvitamin D, insulin-like growth factor 1 (IGF-1), thyroid-stimulating hormone, free thyroxin and intact parathormone. Results: There was a significantly greater increase in serum IGF-1 in G-III than in G II (p < 0.01) or G-I (p < 0.05) during the study period, and a significantly greater increase in plasma tALP in G-III than in G-I (p < 0.05). Serum bALP behavior significantly (p < 0.05) differed between G-II (increase) and G-I (decrease). Plasma TRAP behavior significantly differed between G-II and G-I (p < 0.01) and between G-III and G-II (p < 0.05). Conclusion: Daily dietary intake of hydrolyzed collagen seems to have a potential role in enhancing bone remodeling at key stages of growth and development.

Characterization of Adult Stem/Progenitor Cell Populations From Bone Marrow in a Three-Dimensional Collagen Gel Culture System.

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells which are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and non-adherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4 and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent, hematopoietic, mesenchymal and endothelial specific markers in vitro and can undergo osteogenic differentiation in vivo.

Myocardial angiogenesis induction with bone protein derived growth factors (animal experiment).

Myocardial angiogenesis induction with vascular growth factors constitutes a potential strategy for patients whose coronary artery disease is refractory to conventional treatment. The importance of angiogenesis in bone formation has led to the development of growth factors derived from bovine bone protein. Twelve pigs (mean weight, 73 +/- 3 kg) were chosen for the study. In the first group (n = 6, growth factor group) five 100 micrograms boluses of growth factors derived from bovine bone protein, diluted in Povidone 5%, were injected in the lateral wall of the left ventricle. In the second group (n = 6, control group), the same operation was performed but only the diluting agent was injected. All the animals were sacrificed after 28 days and the vascular density of the left lateral wall (expressed as the number of vascular structures per mm2) as well as the area of blood vessel profiles per myocardial area analysed were determined histologically with a computerised system. The growth factor group had a capillary density which was significantly higher than that of the control group: 12.6 +/- 0.9/mm2 vs 4.8 +/- 0.5/mm2 (p < 0.01). The same holds true for the arteriolar density: 1 +/- 0.2/mm2 vs 0.3 +/- 0.1/mm2 (p < 0.01). The surface ratios of blood vessel profiles per myocardial area were 4900 +/- 800 micron 2/mm2 and 1550 +/- 400 micron 2/mm2 (p < 0.01) respectively. In this experimental model, bovine bone protein derived growth factors induce a significant neovascularisation in healthy myocardium, and appear therefore as promising candidates for therapeutic angiogenesis.

The use of Mycobacterium tuberculosis HspX and GlcB proteins to identify latent tuberculosis in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the destruction of articular cartilage and bone damage. The chronic treatment of RA patients causes a higher susceptibility to infectious diseases such as tuberculosis (TB); one-third of the world’s population is latently infected (LTBI) with Mycobacterium tuberculosis(Mtb). The tuberculin skin test is used to identify individuals LTBI, but many studies have shown that this test is not suitable for RA patients. The goal of this work was to test the specific cellular immune responses to the Mtb malate synthase (GlcB) and heat shock protein X (HspX) antigens of RA patients and to correlate those responses with LTBI status. The T-helper (Th)1, Th17 and Treg-specific immune responses to the GlcB and HspX Mtb antigens were analysed in RA patients candidates for tumour necrosis factor-α blocker treatment. Our results demonstrated that LTBI RA patients had Th1-specific immune responses to GlcB and HspX. Patients were followed up over two years and 14.3% developed active TB. After the development of active TB, RA patients had increased numbers of Th17 and Treg cells, similar to TB patients. These results demonstrate that a GlcB and HspX antigen assay can be used as a diagnostic test to identify LTBI RA patients.

Osteoprotegerin and bone mineral density in hemodiafiltration patients.

A newly identified cytokine, osteoprotegerin (OPG) appears to be involved in the regulation of bone remodeling. In vitro studies suggest that OPG, a soluble member of the TNF receptor family of proteins, inhibits osteoclastogenesis by interrupting the intercellular signaling between osteoblastic stromal cells and osteoclast progenitors. As patients with chronic renal failure (CRF) often have renal osteodystrophy (ROD), we investigated the role of osteoprotegerin (OPG) in ROD, and investigated whether there was any relationship between serum OPG, intact parathyroid (PTH) (iPTH), vitamin D, and trabecular bone. Serum OPG combined with iPTH might be a useful tool in the noninvasive diagnosis of ROD, at least in cases in which the range of PTH values compromises reliable diagnosis. Thirty-six patients on maintenance hemodiafiltration (HDF) and a control group of 36 age and sex matched healthy subjects with no known metabolic bone disease were studied. The following assays were made on serum: iPTH, osteocalcin (BGP), bone alkaline phosphatase, 25(OH)-cholecalciferol, calcium, phosphate, OPG, IGF-1, estradiol, and free testosterone. Serum Ca++, P, B-ALP, BGP, IGF-1, iPTH, and OPG levels were significantly higher in HDF patients than in controls, while DXA measurements and quantitative ultrasound (QUS) parameters were significantly lower. On grouping patients according to their mean OPG levels, we observed significantly lower serum IGF-1, vitamin D3 concentrations, and lumbar spine and hip bone mineral density in the high OPG groups. No correlation was found between OPG and bone turnover markers, whereas a negative correlation was found between serum OPG and IGF-1 levels (r=-0.64, p=0.032). Serum iPTH concentrations were positively correlated with bone alkaline phosphatase (B-ALP) (r=0.69, p=0.038) and BGP (r=0.92, p<0.001). The findings made suggest that an increase in OPG levels may be a compensatory response to elevated bone loss. The low bone mineral density (BMD) levels found in the high OPG group might have been due to the significant decrease in serum IGF-1 and vitamin D3 observed. In conclusion, the findings made in the present study demonstrate that increased OPG in hemodiafiltration patients is only partly due to decreased renal clearance. As it may partly reflect a compensatory response to increased bone loss, this parameter might be helpful in the identification of patients with a marked reduction in trabecular BMD.

PPAR gamma: ally and foe in bone metabolism.

Bone homeostasis is a well-balanced process that is largely dependent on the contribution of both bone-forming osteoblasts and bone-resorbing osteoclasts. A new study (Wan et al., 2007) suggests a previously unsuspected role for the transcription factor PPARgamma in promoting bone progenitors to the osteoclastic lineage.

Rapid death and regeneration of NKT cells in anti-CD3epsilon- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis.

Natural killer T (NKT) cells express a T cell receptor (TCR) and markers common to NK cells, including NK1.1. In vivo, NKT cells are triggered by anti-CD3epsilon MAb to rapidly produce large amounts of IL-4 and by IL-12 to reject tumors. We show here that anti-CD3epsilon MAb treatment rapidly depletes the liver (and partially the spleen) of NKT cells and that homeostasis is achieved 1 to 2 days later via NKT cell proliferation that occurs mainly in bone marrow. Similar results were obtained in mice treated with IL-12. Collectively, our data demonstrate that peripheral NKT cells are highly sensitive to activation-induced cell death and that bone marrow plays a major role in restoring NKT cell homeostasis.

Bone metabolism and risk of secondary hyperparathyroidism 12 months after gastric banding in obese pre-menopausal women.

OBJECTIVE: To evaluate, during the first postoperative year in obese pre-menopausal women, the effects of laparoscopic gastric banding on calcium and vitamin D metabolism, the potential modifications of bone mineral content and bone mineral density, and the risk of development of secondary hyperparathyroidism. SUBJECTS: Thirty-one obese pre-menopausal women aged between 25 and 52 y with a mean body mass index (BMI) of 43.6 kg/m(2), scheduled for gastric banding were included. Patients with renal, hepatic, metabolic and bone disease were excluded. METHODS: Body composition and bone mineral density (BMD) were measured at baseline, 6 and 12 months after gastric banding using dual-energy X-ray absorptiometry. Serum calcium, phosphate, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, bilirubin, urea, creatinine, uric acid, proteins, parathormone, vitamin D(3), IGF-1, IGF-BP3 and telopeptide, as well as urinary telopeptide, were measured at baseline and 1, 3, 6, 9 and 12 months after surgery. RESULTS: After 1 y vitamin D3 remained stable and PTH decreased by 12%, but the difference was not significant. Serum telopeptide C increased significantly by 100% (P<0.001). There was an initial drop of the IGF-BP3 during the first 6 months (P<0.05), but the reduction was no longer significant after 1 y. The BMD of cortical bone (femoral neck) decreased significantly and showed a trend of a positive correlation with the increase of telopeptides (P<0.06). The BMD of trabecular bone, at the lumbar spine, increased proportionally to the reduction of hip circumference and of body fat. CONCLUSION: There is no evidence of secondary hyperparathyroidism 1 y after gastric banding. Nevertheless biochemical bone markers show a negative remodelling balance, characterized by an increase of bone resorption. The serum telopeptide seems to be a reliable parameter, not affected by weight loss, to follow up bone turnover after gastroplasty.

Ultraweak photon emission in assessing bone growth factor efficiency using fibroblastic differentiation.

Photons participate in many atomic and molecular interactions and changes. Recent biophysical research has shown the induction of ultraweak photons in biological tissue. It is now established that plants, animal and human cells emit a very weak radiation which can be readily detected with an appropriate photomultiplier system. Although the emission is extremely low in mammalian cells, it can be efficiently induced by ultraviolet light. In our studies, we used the differentiation system of human skin fibroblasts from a patient with Xeroderma Pigmentosum of complementation group A in order to test the growth stimulation efficiency of various bone growth factors at concentrations as low as 5 ng/ml of cell culture medium. In additional experiments, the cells were irradiated with a moderate fluence of ultraviolet A. The different batches of growth factors showed various proliferation of skin fibroblasts in culture which could be correlated with the ultraweak photon emission. The growth factors reduced the acceleration of the fibroblast differentiation induced by mitomycin C by a factor of 10-30%. In view that fibroblasts play an essential role in skin aging and wound healing, the fibroblast differentiation system is a very useful tool in order to elucidate the efficacy of growth factors.

Molecular pathogenesis of spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins.

The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13(G64D), in which Gly at amino acid position 64 is replaced by Asp, and ZIP13(ΔFLA), which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13(G64D) and ZIP13(ΔFLA) protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

Building bone tissue: matrices and scaffolds in physiology and biotechnology

Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

Bone metabolism and vascular calcification

Osteoporosis and atherosclerosis are chronic degenerative diseases which have been considered to be independent and whose common characteristic is increasing incidence with age. At present, growing evidence indicates the existence of a correlation between cardiovascular disease and osteoporosis, irrespective of age. The morbidity and mortality of osteoporosis is mainly related to the occurrence of fractures. Atherosclerosis shows a high rate of morbidity and especially mortality because of its clinical repercussions such as angina pectoris, acute myocardial infarction, stroke, and peripheral vascular insufficiency. Atherosclerotic disease is characterized by the accumulation of lipid material in the arterial wall resulting from autoimmune and inflammatory mechanisms. More than 90% of these fatty plaques undergo calcification. The correlation between osteoporosis and atherosclerosis is being established by studies of the underlying physiopathological mechanisms, which seem to coincide in many biochemical pathways, and of the risk factors for vascular disease, which have also been associated with a higher incidence of low-bone mineral density. In addition, there is evidence indicating an action of antiresorptive drugs on the reduction of cardiovascular risks and the effect of statins, antihypertensives and insulin on bone mass increase. The mechanism of arterial calcification resembles the process of osteogenesis, involving various cells, proteins and cytokines that lead to tissue mineralization. The authors review the factors responsible for atherosclerotic disease that correlate with low-bone mineral density.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.