Induction of periosteal bone formation by intraosseous BMP-2 injection in a mouse model of osteogenesis imperfecta.

PURPOSE: Surgical interventions are routinely performed on children with osteogenesis imperfecta (OI) to stabilize long bones, often post fracture. We speculated that a combination of intramedullary reaming and intraosseous injection of recombinant bone morphogenetic protein-2 (BMP-2) could enhance periosteal ossification and ultimately cortical thickness and strength. This approach was conceptually tested in a preclinical model of genetic bone fragility. METHODS: Six experimental groups were tested including no treatment, intramedullary reaming, and reaming with 5 µg BMP-2 injection performed in the tibiae of both wild type (WT) and Col1a2 G610C/+ (OI, Amish mutation) mice. Bone formation was examined at a two-week time point in ex vivo specimens by micro-computed tomography (microCT) analysis and histomorphometry with a dynamic bone label. RESULTS: MicroCT data illustrated increases in tibial cortical thickness with intramedullary reaming alone (Saline) and reaming plus BMP-2 injection (BMP-2) compared to no intervention controls. In the OI mice, the periosteal bone increase was not statistically significant with Saline but there was an increase of +192% (p = 0.053) with BMP-2 injection. Dynamic histomorphometry on calcein label was used to quantify new woven bone formation; while BMP-2 induced greater bone formation than Saline, the anabolic response was blunted overall in the OI groups. CONCLUSIONS: These data indicate that targeting the intramedullary compartment via reaming and intraosseous BMP-2 delivery can lead to gains in cortical bone parameters. It is suggested that the next step is to validate safety and functional improvements in a clinical OI setting.

MeCP2 deficiency is associated with reduced levels of tubulin acetylation and can be restored using HDAC6 inhibitors.

UNLABELLED: Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by loss of function mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Despite the genetic cause being known in the majority of cases, the pathophysiology of the neurological phenotype of RTT is largely unknown. Tubulin and the microtubule network play an essential role in neuronal function whereby the acetylation state of microtubules dictates the efficiency of neuronal migration and differentiation, synaptic targeting and molecular motor trafficking of mRNA, high-energy mitochondria and brain-derived neurotrophic factor (BDNF)-containing vesicles. Recent reports have shown perturbations in tubulin and microtubule dynamics in MeCP2-deficient cells, suggesting a link between the aberrations of these cellular entities and the neurobiology of RTT. We have interrogated the functional state of the microtubule network in fibroblasts derived from two patients with RTT as well as cortical neurons from a RTT mouse model and observed a reduction in acetylated α-tubulin and an increase in the tubulin-specific deacetylase, histone deacetylase 6 (HDAC6). Furthermore, we show that inhibition of HDAC6 by Tubastatin A can restore tubulin acetylation levels. We also demonstrate microtubule instability in the RTT patient fibroblasts in response to nocodazole, which is progressively ameliorated in a mutation-dependent manner by Tubastatin A. We conclude that Tubastatin A is capable of counteracting the microtubule defects observed in MeCP2-deficient cells, which could in turn lead to the restoration of molecular trafficking along the microtubules and thus could be a potentially new therapeutic option for RTT. KEY MESSAGE: Cells from MeCP2-deficient cells show reduced levels of acetylated α-tubulin. Cells from two patients and a RTT mouse model have increased levels of HDAC6 but not sirtuin 2 (SIRT2). Inhibition of HDAC6 by Tubastatin A increases the in vitro acetylation of α-tubulin. Inhibition of HDAC6 by Tubastatin A does not increase MECP2 expression. Cells from two patients show microtubule instability, which is ameliorated by Tubastatin A.

Spatial control of bone formation using a porous polymer scaffold co-delivering anabolic rhBMP-2 and anti-resorptive agents.

Current clinical delivery of recombinant human bone morphogenetic proteins (rhBMPs) utilises freeze-dried collagen. Despite effective new bone generation, rhBMP via collagen can be limited by significant complications due to inflammation and uncontrolled bone formation. This study aimed to produce an alternative rhBMP local delivery system to permit more controllable and superior rhBMP-induced bone formation. Cylindrical porous poly(lactic-co-glycolic acid) (PLGA) scaffolds were manufactured by thermally-induced phase separation. Scaffolds were encapsulated with anabolic rhBMP-2 (20 µg) ± anti-resorptive agents: zoledronic acid (5 µg ZA), ZA pre-adsorbed onto hydroxyapatite microparticles, (5 µg ZA/2% HA) or IkappaB kinase (IKK) inhibitor (10 µg PS-1145). Scaffolds were inserted in a 6-mm critical-sized femoral defect in Wistar rats, and compared against rhBMP-2 via collagen. The regenerate region was examined at 6 weeks by 3D microCT and descriptive histology. MicroCT and histology revealed rhBMP-induced bone was more restricted in the PLGA scaffolds than collagen scaffolds (-92.3% TV, p < 0.01). The regenerate formed by PLGA + rhBMP-2/ZA/HA showed comparable bone volume to rhBMP-2 via collagen, and bone mineral density was +9.1% higher (p < 0.01). Local adjunct ZA/HA or PS-1145 significantly enhanced PLGA + rhBMP-induced bone formation by +78.2% and +52.0%, respectively (p ≤ 0.01). Mechanistically, MG-63 human osteoblast-like cells showed cellular invasion and proliferation within PLGA scaffolds. In conclusion, PLGA scaffolds enabled superior spatial control of rhBMP-induced bone formation over clinically-used collagen. The PLGA scaffold has the potential to avoid uncontrollable bone formation-related safety issues and to customise bone shape by scaffold design. Moreover, local treatment with anti-resorptive agents incorporated within the scaffold further augmented rhBMP-induced bone formation.

A sugar-based phase-transitioning delivery system for bone tissue engineering.

Bone tissue engineering approaches commonly involve the delivery of recombinant human bone morphogenetic proteins (rhBMPs). However, there are limitations associated with the currently used carriers, including the need for surgical implantation and the associated increase in infection risk. As an alternative to traditional porous collagen sponge, we have adopted a solution of the injectable sucrose acetate isobutyrate (SAIB) as a carrier for rhBMP-2. The ability to deliver rhBMP-2 and other agents by injection reduces the infection risk and lesion size whilst in surgery, with the potential to avoid open surgery altogether in some indications. The primary methodology used for this in vivo study was a C57BL6/J mouse ectopic bone formation model. Specimens were examined by x-ray, microCT, and histology at 3 weeks. SAIB was delivered non-invasively and produced up to 3-fold greater bone volume compared to collagen. To further refine and improve upon the formulation, SAIB containing rhBMP-2 was admixed with candidate compounds including ceramic microparticles, anti-resorptives, and cell signalling inhibitors and further tested in vivo. The formulation combining SAIB/rhBMP-2, the bisphosphonate zoledronic acid (ZA), and hydroxyapatite (HA) microparticles yielded a 10-fold greater bone volume than SAIB/rhBMP-2 alone. To investigate the mechanism underlying the synergy between ZA and HA, we used in vitro binding assays and in vivo fluorescent biodistribution studies to demonstrate that ceramic particles could bind and sequester the bisphosphonate. These data show the utility of SAIB as a non-invasive rhBMP delivery system as well as describing an optimised formulation for bone tissue engineering.

Effects of chilling on male gametophyte development in rice.

Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic anthers. Anthers observed during tetrad release also have callose (1,3-beta-glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity.

Changes in symplastic permeability during adventitious shoot regeneration in tobacco thin cell layers.

Thin cell layer (TCL) explants of tobacco (Nicotiana tabacum L.) were cultured in either a regeneration medium that resulted in formation of adventitious vegetative shoots or a non-regeneration (control) medium that maintained the TCLs but did not promote shoot formation. Microinjections were conducted on epidermal cells at 1- or 2-day intervals during the culture period (14 days) and also on meristematic regions as they appeared in regenerating TCLs. A fluorescein isothiocyanate-labelled peptide (F(Glu)3 MW 799) was used to assess the permeability of the symplast during adventitious shoot regeneration. A period of increased symplastic movement of F(Glu)3 was detected during day 2 of culture and was significantly greater in regenerating TCLs than in non-regenerating TCLs. This corresponded to the period of the first cell divisions and represents the re-initiation of a meristematic type of symplastic linkage between epidermal cells. A smaller increase in cell-to-cell movement within non-regenerating TCLs indicated a possible stress response as a factor in these changes. Movement of F(Glu)3 throughout the epidermal symplast of regenerating TCLs returned to pre-culture levels by the time of shoot primordia formation. F(Glu)3 movement was further down-regulated in non-regenerating TCLs, with a high degree of cell isolation observed. Within newly formed shoots, symplastic movement of F(Glu)3 cycled between high and low levels.

Cell-to-cell communication via plant endomembranes.

Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue of Nicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans were microinjected using iontophoresis into the cytoplasm andcortical endomembrane network of these cells. The microinjected endomembrane network was similar in location and structure to the endoplasmic reticulum (ER) as revealed by staining with 3, 3'-dihexyloxacarbocyanine iodide (DiOC(6)). No cell-to-cell movement of dextrans was observed following cytoplasmic injections but injection of dextrans into the endomembrane network resulted in rapid diffusion of the probes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellular communication via the desmotubule through plasmodesmata.