Bone Tissue Development Research Articles

Polypeptide growth factors in bone matrix.

The presence of many types of polypeptide growth factors in the mineralized extracellular matrix of bone is now well established. These factors are generally referred to as bone-derived growth factors (BDGFs), and are similar, or possibly identical, to the following species; platelet-derived growth factor (PDGF); acidic and basic forms of fibroblast growth factor (aFGF, bFGF); transforming growth factor beta (TGF-beta); and insulin-like growth factor 1 (IGF-1). Several osteoinductive factors, such as bone morphogenetic protein (BMP) and osteogenin, a skeletal growth factor (SGF), and osteoblast-derived BDGFs, have also been identified. Complete description of the biological functions of these BDGFs which are relevant to bone will ultimately require specific bioassays involving specific cell types in vitro, as well as in vivo animal implant models. Studies with primary rat osteoblast-like cells exposed either to mixed BDGFs, pure TGF-beta, or heparin-purified PDGF, aFGF, or bFGF from bovine bone have shown a general dose-dependent mitogenic effect. Phenotypic changes which accompany the BDGF-induced wave of proliferation include: decreased osteocalcin secretion and a reduction in 1,25-(OH)2 vitamin D3-stimulated osteocalcin synthesis; reduced alkaline phosphatase specific activity; decreased cyclic AMP responsiveness to parathyroid hormone (PTH); and increased collagen synthesis. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than that in serum. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for the unmasking or release of BDGFs from the mineralized matrix that result in local action on osteoblasts, endothelial cells, and other target cells are undoubtedly important for the development and maintenance of bone tissue.

A prospective analysis of the role of silicon in wound care

Silicon is an important micronutrient associated with the development of bone and connective tissue. This article discusses the properties of silicon, its absorption in the human body and its current and potential use in wound management.

Synthesis and characterization of segmented poly(esterurethane urea) elastomers for bone tissue engineering

Segmented polyurethanes have been used extensively in implantable medical devices, but their tunable mechanical properties make them attractive for examining the effect of biomaterial modulus on engineered musculoskeletal tissue development. In this study, a family of segmented degradable poly(esterurethane urea)s (PEUURs) were synthesized from 1,4-diisocyanatobutane, a poly(ε-caprolactone) (PCL) macrodiol soft segment and a tyramine-1,4-diisocyanatobutane-tyramine chain extender. By systematically increasing the PCL macrodiol molecular weight from 1100 to 2700 Da, the storage modulus, crystallinity and melting point of the PCL segment were systematically varied. In particular, the melting temperature, T m, increased from 21 to 61 °C and the storage modulus at 37 °C increased from 52 to 278 MPa with increasing PCL macrodiol molecular weight, suggesting that the crystallinity of the PCL macrodiol contributed significantly to the mechanical properties of the polymers. Bone marrow stromal cells were cultured on rigid polymer films under osteogenic conditions for up to 21 days. Cell density, alkaline phosphatase activity, and osteopontin and osteocalcin expression were similar among PEUURs and comparable to poly( d, l-lactic-coglycolic acid). This study demonstrates the suitability of this family of PEUURs for tissue engineering applications, and establishes a foundation for determining the effect of biomaterial modulus on bone tissue development.

Plasma Concentration of Insulin-like Growth Factor I (IGF-I) in Growing Ardenner Horses Suffering from Juvenile Digital Degenerative Osteoarthropathy

Degenerative osteoarthropathy resulting in a reduced active lifespan was observed in Ardenner horses. In the context of joint biology, insulin-like growth factor I (IGF-I) is a potential candidate to affect the anabolism of cartilage matrix molecules. A group of 30 Ardenner horses reared under standardized conditions from weaning were evaluated periodically from 15 to 28 months of age to detect the early manifestations of the disease. At the end of this period, horses were classified in two pathological groups related to the degree of interphalangeal degenerative osteoarthropathy based on clinical and radiographic evaluations: healthy (46.7%) and pathological (53.3%) horses. Seven sequential blood samples were taken from each horse (during the evaluation period) to study the variation of IGF-I plasma concentration. We tested the variations of the IGF-I plasma concentration during growth, and the effect of sex and of pathological classes. Significant variations were observed during the research period, with a maximum value corresponding to spring and a minimum in autumn. A significant reduction of the IGF-I plasma concentration was also observed in the pathological horses (433.5 +/- 19.5 ng/ml) compared to the healthy horses (493.9 +/- 18.2 ng/ml). An alteration in the level of this growth factor could induce a disregulation of the mechanisms involved in the local control of joint and bone tissue development.

Bone modeling adaptation as a method for promoting development of bone tissue engineered construct in vitro

It was widely accepted that bone adjusts its mass and structure to altered external mechanical environment. In the modeling process, in vivo bone increases bone formation in order to adapt to overload strains. Therefore the bone modeling adaptation as a method may be applied to promote the development of bone tissue engineered construct in vitro. External mechanical force subjected on cell-seeded scaffolds during culture produces overload zone strains in construct, which activates bone cells differentiation and/or extracellular matrix deposition, and promotes bone formation on the interior surface of scaffolds. The mechanical environment enhances the development of bone tissue engineered construct, which recreates the process of bone modeling. Though the overload strains will be restore to the adaptation zone strains due to modeling adaptation, the overload strains made by increasing the amount of external force step by step always remain the bone-forming state of construct. The development of construct will be in progress to accommodate to the new mechanical environment until the bone-like construct is obtained. The method may not only accomplish the bone formation on the base of template (scaffold), but also optimize the mechanical properties of new formation, and finally may produce the off-the-shelf, bone-like substitutes.

The addition of biphasic calcium phosphate to porous chitosan scaffolds enhances bone tissue development in vitro

Uniform distribution of cells and their extracellular matrix is essential for the in vivo success of bone tissue engineering constructs produced in vitro. In this study, the effects of biphasic calcium phosphate (BCP) granules embedded into chitosan scaffolds on the distribution, morphology, and phenotypic expression of osteoblastic cells were investigated. Mesenchymal stem cells (MSCs) and preosteoblasts were cultured on chitosan scaffolds with and without BCP under osteoblastic differentiation/maturation conditions for periods up to 4 weeks. The addition of 25 wt % BCP to chitosan created a uniform layer of calcium phosphate (CaP) precipitation similar to bone mineral on the scaffold surfaces as determined by scanning electron microscopy and X-ray spectroscopy. Scaffolds with this CaP layer yielded more uniform and complete cell and ECM distribution than chitosan scaffolds without BCP. The suggestion of chemotaxis in the appearance of this response was confirmed by successive experiments in a Boyden chamber. The CaP layer also altered morphology of cells initially attached to the scaffold surfaces, leading to higher expression of marker proteins of osteoblastic phenotype including alkaline phosphatase and osteocalcin. The use of chitosan/BCP scaffolds for culture of MSCs and preosteoblasts enhances bone tissue development in vitro.

Regulation of Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells by Laminin‐5 (Laminin‐332)

Laminin-5 (laminin-332) is an important basement membrane protein that regulates cell attachment and motility. Recent studies have shown that laminin-5 is expressed in human mesenchymal stem cells (MSCs) in culture and that the laminin gamma2 chain is transiently expressed in chondrocytes during development. These studies suggest that laminin-5 may be involved in the regulation of chondrogenic differentiation of MSCs. In this study, we examined a possible role of laminin-5 in the proliferation and differentiation of human MSCs. When MSCs were incubated in the presence of a coated or soluble form of laminin-5 in a growth medium, they proliferated more rapidly than nontreated cells, keeping their differentiation potential. On the other hand, laminin-5 potently suppressed the chondrogenic differentiation of MSCs. These activities were mediated mainly by integrin alpha3beta1. However, laminin-5 had no effect on the osteogenic differentiation of MSCs. These results suggest that laminin-5 may contribute to the development of bone tissues by promoting the proliferation and by suppressing the chondrogenic differentiation of MSCs.

MODULATING EFFECTS OF IGF I AND LEPTIN LEVELS ON BONE MINERAL CONTENT

Knowledge the strong relationship between body lipid size and serum leptin levels due to its appetite regulation and the insulin like growth factor (IGF) I involved as a signal of calories, body lipid composition and proteins viability, it is important to begin to answer questions concerning bone formation throughout leptin and IGF I receptors. The aim of this study was to evaluate the strong association among leptin and IGF I levels during active growth. Forty two weanling Wistar rats were randomly assigned one of three diets providing a carbohydrate to lipid calorie ratio of 1:1 (reference group) (a) or 3:1, with either 0.6% or 0.3% of calcium content (b and c, respectively). At age= 35 d, animals were sacrificed. Body weights (BW=g) and lengths (BL=cm) were measured and body composition (% body fat by chemical method AOAC) and bone mineral content (BMC) (DEXA Lunar DPX) were determined. In five of them serum IGF I and leptin levels were measured (ELISA ng/ml). Results as mean ± SD. Diets in which lipids were restricted (b and c), affected the % of body fat and bone mineral content. Body length remained unchanged in adequate calcium diets while there was a tendency towards serum IGF I and leptin levels more diminished in b and c, than a. Serum IGF I and leptin appear to account for an accurate bone tissue development. Awarded by UBACyT O 004.

A Polymorphism in a Conserved Posttranscriptional Regulatory Motif Alters Bone Morphogenetic Protein 2 (BMP2) RNA:Protein Interactions

The bone morphogenetic protein (BMP)2 gene has been genetically linked to osteoporosis and osteoarthritis. We have shown that the 3'-untranslated regions (UTR) of BMP2 genes from mammals to fishes are extraordinarily conserved. This indicates that the BMP2 3'-UTR is under stringent selective pressure. We present evidence that the conserved region is a strong posttranscriptional regulator of BMP2 expression. Polymorphisms in cis-regulatory elements have been proven to influence susceptibility to a growing number of diseases. A common single nucleotide polymorphism (SNP) disrupts a putative posttranscriptional regulatory motif, an AU-rich element, within the BMP2 3'-UTR. The affinity of specific proteins for the rs15705 SNP sequence differs from their affinity for the normal human sequence. More importantly, the in vitro decay rate of RNAs with the SNP is higher than that of RNAs with the normal sequence. Such changes in mRNA:protein interactions may influence the posttranscriptional mechanisms that control BMP2 gene expression. The consequent alterations in BMP2 protein levels may influence the development or physiology of bone or other BMP2-influenced tissues.

Body tissue development in lambs of two genetic lines analysed by X-ray computer tomography

Growth and development of carcass lean (LEAN), carcass fat (FAT), BONE, subcutaneous fat (SFAT), intermuscular fat (IeFAT), non-carcass fat (NcFAT) and non-carcass visceral tissue (NcVT) were analysed by computer tomography (CT) in male lambs of a meat line (ML, n = 62) and a control line of sheep (CL, n = 29). The ML animals were of origin Norwegian white sheep (NWS) crossed with Texel and selected for lean weight since 2001, while the CL was NWS animals bred for an overall national breeding goal. CT images were recorded at 40 mm intervals throughout the animal at a mean age of 44 (CT1), 90 (CT2) and 118 (CT3) days. Weight of body tissues was estimated by CT in each animal for each scanning. Traits were analysed by mixed model including line, dam age, year, age at CT1 (covariate) and random effect of lamb and scan event (lamb). Average daily growth (g/day) was analysed by general linear model in SAS including fixed effects and their two-traits interactions and age at CT1 (covariate). ML lambs had a heavier weight of LEAN at CT1 ( P < 0.05) and showed a tendency of more LEAN at CT2 and CT3 than the CL. LEAN:BONE was significant larger for the ML compared to CL at all scan events, while FAT, FAT% and LEAN:FAT were similar for the two lines for all scan events ( P > 0.05). CL had a greater average daily growth between the second and third scan event than ML but this was largely a larger growth in fat depots.

SWI/SNF chromatin remodeling complex is obligatory for BMP2‐induced, Runx2‐dependent skeletal gene expression that controls osteoblast differentiation

Development of bone tissue requires maturation of osteoblasts from mesenchymal precursors. BMP2, a member of the TGFbeta superfamily, and the Runx2 (AML3/Cbfa1) transcription factor, a downstream BMP2 effector, are regulatory signals required for osteoblast differentiation. While Runx2 responsive osteogenic gene expression has been functionally linked to alterations in chromatin structure, the factors that govern this chromatin remodeling remain to be identified. Here, we address the role of the SWI/SNF chromatin remodeling enzymes in BMP2-induced, Runx2-dependent development of the osteoblast phenotype. For these studies, we have examined calvarial cells from wild-type (WT) mice and mice that are homozygous for the Runx2 null allele, as well as the C2C12 model of BMP2-induced osteogenesis. By the analysis of microarray data, we find that several components of the SWI/SNF complex are regulated during BMP2-mediated osteoblast differentiation. Brg1 is an essential DNA dependent ATPase subunit of the SWI/SNF complex. Thus, functional studies were carried out using a fibroblast cell line that conditionally expresses a mutant Brg1 protein, which exerts a dominant negative effect on SWI/SNF function. Our findings demonstrate that SWI/SNF is required for BMP2-induced expression of alkaline phosphatase (APase), an early marker reflecting Runx2 control of osteoblast differentiation. In addition, Brg1 is expressed in cells within the developing skeleton of the mouse embryo as well as in osteoblasts ex vivo. Taken together these results support the concept that BMP2-mediated osteogenesis requires Runx2, and demonstrates that initiation of BMP2-induced, Runx2-dependent skeletal gene expression requires SWI/SNF chromatin remodeling complexes.

Titanium oxide nanotubes for bone regeneration.

Titanium oxide nanotubes with Ca ions on their surfaces were prepared as 2 mm cylindrical inserts and placed into surgically created bone defects in the femurs of Wistar rats. On day 3, fibroblast-like cells were present on the surface of the nanotube inserts and fibers were observed by scanning electron microscopy (SEM). On day 7, cells with alkaline phosphatase activity were present and identified as osteoblasts by SEM and transmission electron microscopy. New bone matrices were observed in and around the porous nanotube inserts by light microscopy. Compared with clinically used hydroxyapatite and tricalcium phosphate, beta-titanium oxide nanotubes promote faster acquisition and development of osteoblasts and bone tissues and have better bone regenerating ability after one week.

Lymphocyte development and function in the absence of retinoic acid-related orphan receptor alpha.

The orphan nuclear receptor, retinoid acid-related orphan receptor (ROR)alpha, is essential for the development of cerebellar Purkinje cells and bone tissue. RORalpha may also play a critical role in lymphocyte development and function because staggerer mice, a natural mutant strain with a disrupted expression of RORalpha, have reduced thymic and splenic cellularity. In this report, we analyzed the role of RORalpha in lymphocyte development by examining lymphoid compartments in RORalpha(-/-) mice and Rag-2(-/-) mice reconstituted with RORalpha(-/-) bone marrow. We found that T and B cell development was severely defective in RORalpha(-/-) mice, but not in Rag-2(-/-)/RORalpha(-/-) chimeric mice. We also analyzed cellular and humoral immune responses in Rag-2(-/-)/RORalpha(-/-) chimeric mice. Our results show that serum IgG levels were elevated in Rag-2(-/-)/RORalpha(-/-) chimeric mice after immunization with a T-dependent Ag compared with control chimeras. IFN-gamma production by RORalpha(-/-) CD8(+) T cells after TCR stimulation was also increased. Furthermore, RORalpha(-/-) mast cells and macrophages produced an increased amount of TNF-alpha and IL-6 upon activation. These results indicate that RORalpha indirectly regulates lymphocyte development by providing an appropriate microenvironment and controls immune responses by negatively regulating cytokine production in innate immune cells and lymphocytes.

Hydrodynamic shear stimulates osteocalcin expression but not proliferation of bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are a promising component for engineered bone tissues, but in vitro formation of a bonelike tissue requires culture conditions that direct these multipotent cells toward osteoblastic maturation. Fluid flow has been postulated to stimulate bone tissue development in vivo, but the effect of shear stress on proliferation and differentiation of osteoprogenitor cell cultures in vitro has not been examined closely. In this study BMSCs were cultured on fibronectin-coated substrates and exposed intermittently (for 30 min 3, 5, 7, 9, 11, and 13 days after seeding) to a spatially dependent range of shear stresses (0.36 to 2.7 dyn/cm(2)) using a radial-flow chamber. After 7 days cell density did not vary between sheared and control cell layers. In contrast, after 21 days the accumulation of osteocalcin protein (OC) in cell layers was increased significantly relative to static controls, while the quantity of multilayer cell aggregates (i.e., bone nodules) was diminished. Neither of these effects varied systematically with shear magnitude. Finally, pretreatment of cultures with the cyclooxygenase (COX)-2-specific inhibitor NS-398 blocked prostaglandin secretion in response to shearing flow and significantly reduced OC accumulation in cell layers. These results provide evidence that flow stimulates osteoblastic maturation but not proliferation of bone marrow stromal cells and that prostaglandin signaling is involved in this effect.

The silicon content of beer and its bioavailability in healthy volunteers.

Dietary Si, as soluble orthosilicic acid (OSA), may be important for the growth and development of bone and connective tissue. Beer appears to be a major contributor to Si intake, although the Si content of beer and its bioavailability in human subjects have not been well established. Here we investigated the Si content of different beers and then estimated Si absorption from beer in healthy volunteers. The Si content of seventy-six different beers was estimated using inductively coupled plasma optical emission spectrometry and one of the beers, used in the ingestion study, was ultrafiltered to determine OSA content. Next, following the ingestion of 0.6 litres beer (22.5 mg Si; 4.6 % (v/v) ethanol), serum and urinary Si levels were measured in nine healthy volunteers over a 6 h period. A solution of OSA was similarly investigated as a positive control and water and 4.6 % ethanol as negative controls. The mean Si level of beer was 19.2 (sd 6.6) mg/l; the median Si level was 18.0 mg/l. There was no significant difference in the Si levels of the different beers by geographical origin or type of beer. Serum and urinary Si levels increased considerably following the ingestion of beer or a solution of OSA but not with the ingestion of either 4.6 % ethanol or water. The ultrafilterability of Si from beer (about 80 %) and its absorption in volunteers (about 55 %) was comparable with that of a solution of OSA suggesting that Si in beer is present chiefly in a monomeric form and is readily bioavailable.

Regulating bone formation via controlled scaffold degradation.

It is widely assumed that coupling the degradation rate of polymers used as cell transplantation carriers to the growth rate of the developing tissue will improve its quantity or quality. To test this hypothesis, we developed alginate hydrogels with a range of degradation rates by gamma-irradiating high-molecular-weight alginate to yield polymers of various molecular weights and structures. Decreasing the size of the polymer chains increased the degradation rate in vivo, as measured by implant retrieval rates, masses, and elastic moduli. Rapidly and slowly degrading alginates, covalently modified with RGD-containing peptides to control cell behavior, were then used to investigate the effect of biodegradation rate on bone tissue development in vivo. The more rapidly degrading gels led to dramatic increases in the extent and quality of bone formation. These results indicate that biomaterial degradability is a critical design criterion for achieving optimal tissue regeneration with cell transplantation.

Biomechanical strategies for articular cartilage regeneration.

Two major contributions to the development of articular cartilage are growth factors and mechanical loading. Growth factors have long been used to modulate the secretion of certain molecules from different cells. The TGF-beta superfamily, specifically the BMPs, CDMPs, OPs, and GDFs, have a dramatic effect on the development of bone and cartilage tissue. These growth factors help produce an extracellular matrix that can withstand extreme loading conditions in the body. In addition to growth factors, it is known that mechanical forces stimulate the synthesis of extracellular proteins in vitro and in vivo and can affect the tissue's overall structure. Load-bearing tissue, such as articular cartilage, will atrophy in the absence of mechanical forces, and this observation has caused researchers to incorporate mechanical stimulation into the tissue engineering process. This article focuses on the importance of mechanical forces in tissue engineering of articular cartilage and the growth factors that help stimulate the formation of load-bearing tissue.

The incidence of injuries in elite junior figure skaters.

There has been rapid growth in the technical and physiologic demands made on skaters who perform more and more difficult jumps, spins, lifts, throws, and free skating movements. To investigate the frequency of injuries and overuse syndromes in elite junior skaters. Questionnaire. During four consecutive Junior World Figure Skating Championships and the Croatia Cup, we interviewed 236 female and 233 male skaters by questionnaire to determine the frequency of injuries and overuse syndromes. Fifty-nine of the female skaters (25%) and 65 of the male skaters (27.9%) reported sustaining acute injuries; 101 female (42.8%) and 106 male (45.5%) skaters reported overuse syndromes. Low back pain was reported by 19 female and 23 male skaters. The most frequent acute injury was ankle sprain. In singles female skaters, the most frequent overuse injury was stress fracture (19.8%), followed by jumper's knee (14.9%). In singles male skaters, jumper's knee (16.1%) was the most frequent injury, followed by Osgood-Schlatter disease (14.2%). More than 50% of injuries in young singles figure skaters involved overuse syndromes. Pairs skaters and ice dance skaters had a higher risk of acute injury than overuse syndrome because of falls from lifts and throw jumps. Programs to improve postural alignment, flexibility, and strength, especially during the asynchronous period of bone and soft tissue development, should be instituted to prevent and reduce overuse syndromes.

Physiological responses of osteoblasts to cyclic stretching and the change of intracellular calcium concentration

The development of bone tissues is regulated by mechanical stimulation. Cyclic stretching was applied to the osteoblasts that were delivered from rat calvarie. The results showed that stretching at 500 μe increased the cell proliferation while loading at 1000 μe and 1500 μe inhabited cell growth. Loading also increased the adhesive force between cells and substrate as well as spreading areas of osteobalsts. Furthermore, the fluorescence probe Fluo-3/AM was used to investigate the effect of stretching stimulation on the intracellular calcium concentration of osteoblasts. The intracellular calcium concentration of osteoblasts that were stretched at 500 μe for 5 min was 92.9% higher than the control. After being treated with the panax ontoginseng saponins, the stretched osteoblasts still expressed 28.6% higher intracellular calcium concentration than that of the control, which proved that both the influx of extracellular calcium and the release of intracellular calcium store were involved in the increase of intracellular calcium concentration when osteoblasts responded to the cyclic stretching. And the influx of extracellular calcium through transmembrance channel played a main role.

Practical Modeling Concepts for Connective Tissue Stem Cell and Progenitor Compartment Kinetics.

Stem cell activation and development is central to skeletal development, maintenance, and repair, as it is for all tissues. However, an integrated model of stem cell proliferation, differentiation, and transit between functional compartments has yet to evolve. In this paper, the authors review current concepts in stem cell biology and progenitor cell growth and differentiation kinetics in the context of bone formation. A cell-based modeling strategy is developed and offered as a tool for conceptual and quantitative exploration of the key kinetic variables and possible organizational hierarchies in bone tissue development and remodeling, as well as in tissue engineering strategies for bone repair.