The Structure of Mineralized Collagen Fibrils

A new model for the structure of mineralized bone collagen is presented which is compatible with neutron diffraction, electronmicroscopic, crosslinking, and composition-density data. Mineralized collagen fibrils are comprised of azimuthally oriented, flexible molecules laterally arranged on a superlattice. Four nearest neighbors are longitudinally staggered by 67 nm and two neighbors by 2* 67 nm. In early stages of mineralization the molecules are parallel to the fibril axis with an average interaxis distance of 1.8 nm. In later stages they become flexed away from the fibril axis by an anisotropic lateral compression of molecules to an interaxis distance of 1.3 nm. Three quarters of the mineral in bone is disposed within the fibrils with a symetry and habit reflecting the above organization of the collagen molecules.     

Vascular Endothelial Growth Pattern During Demineralized Bone Matrix Induced Osteogenesis

The purpose of the current study was to determine the timely ingrowth of blood vessels associated with demineralized bone matrix (DMB) induced osteogenesis. Critical-size (10 × 5mm), full thickness bony defects in rabbit parietal bone were implanted with DBM. Histological and ultrastructural changes were examined 1, 2, 3, 4, 5, 6, 7 and 14 days later. Neovascularization was assessed by immunohistochemical staining for factor VIII antigen (marker for vascular endothelium) and also confirmed by staining using pan-endothelial antibody (CD31) (a marker for endothelium). Immunohistochemical evaluation revealed a positive staining for CD31 and Factor VIII expressed by endothelial cells by day 3 post grafting. By day 4, small blood vessels were first seen budding from host bed towards the grafted DBM. Ultrastructural identification of cells in the early stages of healing revealed the presence of macrophages. The monocyte-derived macrophage appears to play a central role in the repair process using DBM. Results of this study demonstrated a rapid vascularization during the DBM induced osteogenesis. This rapid vascularization is vital to the healing and bone induction ability of the DBM.     

Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population

Using animal models for autoimmune diseases, we have previously shown that allogeneic bone marrow transplantation (allo BMT) can be used to treat autoimmune diseases. Using cynomolgus monkeys, we have recently developed new BMT methods for the treatment of autoimmune diseases. The methods include the perfusion method (PM) for the collection of bone marrow cells (BMCs), and intra-bone marrow (IBM)-BMT for the direct injection of collected whole BMCs into the bone marrow cavity. The PM, in comparison with the conventional aspiration method, can minimize the contamination of BMCs with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease (GvHD) develops in the case of the PM. Since BMCs collected by the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM–BMT) facilitates the engraftment of donor hemopoietic cells. In organ allografts with IBM–BMT, no graft failure occurs even if the radiation dose is reduced. In addition, IBM–BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, since it can efficiently recruit donor-derived normal MSCs.

We have also found that IBM–BMT in conjunction with donor lymphocyte infusion can prevent GvHD, but suppress tumor growth. We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ allografts) and regeneration therapy.     

A general IGF-I overexpression effectively rescued somatic growth and bone deficiency in mice caused by growth hormone receptor knockout

Both growth hormone and insulin-like growth factor (IGF)-I are essential for postnatal somatic growth, while exerting distinct effects on energy homeostasis. Although growth hormone controls IGF-I production, whether IGF-I was the exclusive mediator of its growth promotion is still debated. In order to further explore their in vivo interactions in somatic growth as well as in energy homeostasis, we have crossed mutant (MT-IGF) transgenic mice onto the GHR ? / ? background. As expected, GHR gene deficiency caused growth retardation, including significant decreases in lumbar, femur and total body lengths, as well as decreased bone area, mineral content and mineral density. IGF-I overexpression alone in MT-IGF mice increased the weight, with no significant change in bone mineralization or longitudinal growth. Compared to GHR ? / ? littermates, overexpressed IGF-I in bitransgenic mice (GHR ? / ? and MT-IGF positive) exhibited fully restored body weight, lumbar (but not femur) and total body lengths, and normalized overall bone area, mineral content and density. On the other hand, there were significant changes in fasting glucose level, glucose tolerance, lean/fat masses and even adipose histology as a result of the transgenic/knockout double-crossing. IGF-I overexpression normalized glucose tolerance in GHR ? / ? mice. Intriguingly, on GHR+/ ? background of partial growth hormone insensitivity, overexpression of IGF-I caused a significant weight gain. Our results thus establish that the growth defect and bone deficiency caused by lack of growth hormone signaling can be effectively restored by increasing IGF-I production in vivo.     

Exogenously added BMP-6, BMP-7 and VEGF may not enhance the osteogenic differentiation of human adipose stem cells

Abstract

In the present study bone morphogenetic protein (BMP)-6 alone or in synergy with BMP-7 and vascular endothelial growth factor (VEGF) were tested with human adipose stem cells (hASCs) seeded on cell culture plastic or 3D bioactive glass. Osteogenic medium (OM) was used as a positive control for osteogenic differentiation. The same growth factor groups were also tested combined with OM. None of the growth factor treatments could enhance the osteogenic differentiation of hASCs in 3D- or 2D-culture compared to control or OM. In 3D-culture OM promoted significantly total collagen production, whereas in 2D-culture OM induced high total ALP activity and mineralization compared to control and growth factors groups, but also high cell proliferation. In this study, hASCs did not respond to exogenously added growth although various parameters of the study set-up may have affected these findings contradictory to the previous literature.     

Long-term effects for acute phase myocardial infarct VEGF165 gene transfer cardiac extracellular matrix remodeling

Background. Cardiac remodeling is ultimately regulated by components of the extracellular matrix (ECM). We investigated the important role that growth factors play in the regulation of ECM remodeling that occurs as a consequence of myocardium damage.

Methods and results. Rats were submitted to the ligation of the left anterior coronary artery and pcDNA3-vascular endothelial growth factor (VEGF)₁₆₅ was immediately injected intramyocardially in the treated group. The animals were divided into large size myocardium infarction (LMI) and small size myocardium infarction, with or without gene transfer. The plasmid-containing DNA encoding VEGF₁₆₅ was injected into the cardiac muscle and its effect was observed on the ECM components. Glycosaminoglycans were identified and quantified by agarose gel based electrophoresis and ELISA as well as immunocytochemistry to examine specific cathepsin B, heparanase, and syndecan-4 changes. The amounts of hyaluronic acid (HA; p < 0.005), DS, chondroitin sulfate, and heparan sulfate (p < 0.001) were significantly increased in the LMI treated group in comparison to the other groups, which correlates with the decrease in the expression of heparanase. A decrease in the molecular mass of HA was found in the scar tissue of treated group.

Conclusions. The data obtained strongly support the idea that changes in the ECM and its components are important determinants of cardiac remodeling after myocardium infarct and may be essential for inflammatory response and attempt to stabilize the damage and provide a compensatory mechanisms to maintain cardiac output since the ECM components analyzed are involved with angiogenesis, cell proliferation and differentiation.     

Comparison of platelet-rich plasma,bovine BMP,and rhBMP-4 on bone matrix protein expression in vitro

This study investigated the potential use of platelet-rich plasma (PRP) in conjunction with mRNA expression of bone matrix proteins using bioassay and RT-PCR comparing bovine bone morphogenetic proteins (BMP), recombinant human BMP-4 (rhBMP-4) during rat bone marrow stromal cell (Mesenchymal Stem Cell) differentiation at 14 days. The results showed that all three growth factors were associated with significantly elevated alkaline phosphatase activity. PRP and bovine BMP resulted in increased protein content. The mRNA of type I collagen was expressed with all three growth factors and remained consistently elevated. Osteopontin was observed with PRP from days 1 to 7; bone sialoprotein expression was detected on days 1 and 3. PRP, bovine BMP and rhBMP-4 enhanced the steady-state expression of PDGF-A as time-dependent to day 14 and in PRP was the strongest. PTHr was expressed at days 1 and 5. Vascular endothelial growth factor expression was the most highly expressed after day 3. These findings suggest that PRP increases mRNA expression of bone matrix protein, enchances osteogenesis and angiogenesis in vitro.     

The Effect of Poly(d,l-Lactide-co-Glycolide)-Alendronate Conjugate Nanoparticles on Human Osteoclast Precursors

Abstract

Nanoparticles (NPs) formed from polymers conjugated with bisphosphonates (BPs) allow the bone targeting of loaded drugs, such as doxorubicin, for the treatment of skeletal tumours. The additional antiosteoclastic effect of the conjugated BP could contribute to the inhibition of tumour-associated bone degradation. With this aim, we have produced NPs made of poly(d,l-lactide-co-glycolide) (PLGA) conjugated with alendronate (ALE). To show if ALE retained the antiosteoclastic properties after the conjugation with PLGA and the production of NPs, we treated human osteoclasts, derived from circulating precursors, with PLGA–ALE NPs and compared the effects on actin ring generation, apoptosis and type-I collagen degradation with those of free ALE and with NPs made of pure PLGA. PLGA–ALE NPs disrupted actin ring, induced apoptosis and inhibited collagen degradation. Unexpectedly, also NPs made of pure PLGA showed similar effects. Therefore, we cannot exclude that in addition to the observed antiosteoclastic activity dependent on ALE in PLGA–ALE NPs, there was also an effect due to pure PLGA. Still, as PLGA–ALE NPs are intended for the loading with drugs for the treatment of osteolytic bone metastases, the additional antiosteoclastic effect of PLGA–ALE NPs, and even of PLGA, may contribute to the inhibition of the disease-associated bone degradation.     

Development of chitosan/gelatin hydrogels incorporation of biphasic calcium phosphate nanoparticles for bone tissue engineering

Abstract

The chitosan/gelatin hydrogel incorporated with biphasic calcium phosphate nanoparticles (BCP-NPs) as scaffold (CGB) for bone tissue engineering was reported in this article. Such nanocomposite hydrogels were fabricated by using cycled freeze-thawing method, of which physicochemical and biological properties were regulated by adjusting the weight ratio of chitosan/gelatin/BCP-NPs. The needle-like BCP-NPs were dispersed into composites uniformly, and physically cross-linked with chitosan and gelatin, which were identified via Scanning Electron Microscope (SEM) images and Fourier Transform Infrared Spectroscopy (FT-IR) analysis. The porosity, equilibrium swelling ratio, and compressive strength of CGB scaffolds were mainly influenced by the BCP-NPs concentration. In vitro degradation analysis in simulated body fluids (SBF) displayed that CGB scaffolds were degraded up to at least 30?wt% in one month. Also, CCK-8 analysis confirmed that the prepared scaffolds had a good cytocompatibility through in culturing with bone marrow mesenchymal stem cells (BMSCs). Finally, In vivo animal experiments revealed that new bone tissue was observed inside the scaffolds, and gradually increased with increasing months, when implanted CGB scaffolds into large necrotic lesions of rabbit femoral head. The above results suggested that prepared CGB nanocomposites had the potential to be applied in bone tissue engineering.