Open Access

The need for bone grafts is high, due to age-related diseases, such as tumor resections, but also accidents, risky sports, and military conflicts. The gold standard for bone grafting is the use of autografts from the iliac crest, but the limited amount of accessible material demands new sources of bone replacement. The use of mesenchymal stem cells or their descendant cells, namely osteoblast, the bone-building cells and endothelial cells for angiogenesis, combined with artificial scaffolds, is a new approach. Mesenchymal stem cells (MSCs) can be obtained from the patient themselves, or from donors, as they barely cause an immune response in the recipient. However, MSCs never fully differentiate in vitro which might lead to unwanted effects in vivo. Interestingly, purinergic receptors can positively influence the differentiation of both osteoblasts and endothelial cells, using specific artificial ligands. An overview is given on purinergic receptor signaling in the most-needed cell types involved in bone metabolism-namely osteoblasts, osteoclasts, and endothelial cells. Furthermore, different types of scaffolds and their production methods will be elucidated. Finally, recent patents on scaffold materials, as wells as purinergic receptor-influencing molecules which might impact bone grafting, are discussed.

Echinoderm skeletons are described within a hierarchical framework ranging from complete organisms to the ultrastructural level. They consist of numerous elements which can be isolated, connected by soft tissue or locked together in rigid structures. The top level considers skeletons as a whole with all associated elements and the basic symmetry of the echinoderms. The next level deals with the structural analysis and modeling of echinoids with respect to the growth parameters and stress resistance of the corona. The flexibility and movement has also been studied for the stalks and arms of recent and fossil crinoids. The next level deals with the elaborate morphology and types of symmetry found in single skeletal elements. The numerous types of stereom architectures found within the elements of all echinoderms are highly correlated to specific functions. A high number of recent studies concern the last hierarchical level on ultrastructure and biomineralization. Lightweight aspects of the skeleton are especially present at the level of conjoined plates, single elements and the stereom.

Human hereditary hyperekplexia ("startle disease") is a neurological disorder characterized by exaggerated, convulsive movements in response to unexpected stimuli. Molecular genetic studies have shown that this disease is often caused by amino acid substitutions at arginine 271 to glutamine or leucine of the alpha1 subunit of the inhibitory glycine receptor (GlyR). When exogenously expressed in Xenopus oocytes, agonist responses of mutant alpha1(R271Q) and alpha1(R271L) GlyRs show higher EC50 values and lower maximal inducible responses (relative efficacies) compared with oocytes expressing wild-type alpha1 GlyR subunits. Here, we report that the maximal glycine-induced currents (I(max)) of mutant alpha1(R271Q) and alpha1(R271L) GlyRs were dramatically potentiated in the presence of the anesthetic propofol (PRO), whereas the I(max) of wild-type alpha(1) receptors was not affected. Quantitative analysis of the agonist responses of the isofunctionally substituted alpha1(R271K) mutant GlyR revealed that saturating concentrations of PRO decreased the EC50 values of both glycine and the partial agonist beta-alanine by >10-fold, with relative efficacies increasing by 4- and 16-fold, respectively. Transgenic (tg) mice carrying the alpha1(R271Q) mutation (tg271Q-300) have both spontaneous and induced tremor episodes that closely resemble the movements of startled hyperekplexic patients. After treatment with subanesthetic doses of PRO, the tg271Q-300 mutant mice showed temporary reflexive and locomotor improvements that made them indistinguishable from wild-type mice. Together, these results demonstrate that the functional and behavioral effects of hyperekplexia mutations can be effectively reversed by drugs that potentiate GlyR responses.

Hereditary hyperekplexia is caused by disinhibition of motoneurons resulting from mutations in the ionotropic receptor for the inhibitory neurotransmitter glycine (GlyR). To study the pathomechanisms involved in vivo, we generated and analyzed transgenic mice expressing the hyperekplexia-specific dominant mutant human GlyR alpha1 subunit 271Q. Tg271Q transgenic mice, in contrast to transgenic animals expressing a wild-type human alpha1 subunit (tg271R), display a dramatic phenotype similar to spontaneous and engineered mouse mutations expressing reduced levels of GlyR. Electrophysiological analysis in the ventral horn of the spinal cord of tg271Q mice revealed a diminished GlyR transmission. Intriguingly, an even larger reduction was found for GABA(A)-receptor-mediated inhibitory transmission, indicating that the expression of this disease gene not only affects the glycinergic system but also leads to a drastic downregulation of the entire postsynaptic inhibition. Therefore, the transgenic mice generated here provide a new animal model of systemic receptor interaction to study inherited and acquired neuromotor deficiencies at different functional levels and to develop novel therapeutic concepts for these diseases.

Ultrastructural investigations were performed in young (approximately 2 months) and old (7 months) Mpv17-negative and wild-type mice. The onset, the severity and the pattern of the degeneration significantly differed between both mice strains. In the wild-type mouse strain the degenerative changes of the cochlear structures were similar to the aging pattern described for other species. In contrast, the Mpv17 mutants showed degenerative changes of the cochlear structures already at the age of 2 months. The degenerative changes were patchy arranged throughout the entire length of the cochlea and involved the organ of Corti as well as the stria vascularis epithelia with alterations of the basement membrane of the capillaries. The severe sensorineural hearing loss and degenerative changes of the cochlear structures indicate that cochlear structures, especially the outer hair cells and the intermediate cells of the stria vascularis, are vulnerable to the missing Mpv17 gene product.

Startle disease or hereditary hyperekplexia has been shown to result from mutations in the alpha1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR alpha or beta-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR beta transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR beta-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR beta transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at approximately 3-4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease.

The mutant mouse strain Mpv17-/-, carries a retroviral germline integration that inactivates the Mpv17 gene. Mpv17-deficient mice develop progressive glomerulosclerosis and sensineural deafness at early age. Characteristic basement membrane alterations are found in both sites of pathology. Mpv17 is a peroxisomal protein involved in the metabolism of reactive oxygen species, yet its molecular function is unknown. Dysregulation of antioxidant enzymes and basal membrane components has been established in this model and successful therapeutic intervention with antioxidants prove the causal role of reactive oxygen species in the development of the disease phenotype. We here investigated if the Mpv17-/- mice might be hypertensive. Indeed, our study revealed that Mpv17-/- mice developed significant systemic hypertension and tachycardia between 4 weeks and 5 months of age, accompanied by polyuria and elevated natriuresis. Judging from serum and urine parameters, the hypertensive condition develops concomitantly with the renal disease. Biochemical and pharmacological studies that used the endothelin receptor antagonist bosentan and the angiotensin converting enzyme inhibitor cilazapril indicated no involvement of the endothelin and renin-angiotensin systems in this hypertension, suggesting a potential novel mechanism of blood pressure regulation in this new murine hypertension model. Thus, Mpv17-/- mice unravel an intriguing new association between a defect in reactive oxygen metabolism and the age-dependent development of hypertension.

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

The Mpv 20 transgenic mouse strain was created by infection of embryos with a defective retrovirus. When Mpv 20 heterozygous animals were crossed, no homozygous neonatal mice or midgestation embryos were identified. When embryos from heterozygous crosses were cultured in vitro, approximately one quarter arrested as uncompacted eight-cell embryos, indicating that proviral insertion resulted in a recessive lethal defect whose phenotype was manifest very early in development. Molecular cloning of the Mpv 20 insertion site revealed that the provirus had disrupted the Npat gene, a gene of unknown function, resulting in the production of a truncated Npat mRNA. Expression of the closely linked Atm gene was found to be unaffected by the provirus.

Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses.

We determined the expression pattern of the matrix metalloproteinase interstitial collagenase (MMP-1) during mouse embryo development using in situ hybridization and immunohistochemistry. Localized MMP-1 mRNA was first detected at 14.5 days postconceptus. The spatial and temporal expression was restricted to areas of endochondral and intramembranous bone formation, such as in the mandibula, maxilla, clavicle, scapula, in the vertebrae, and in the dorsal, but not the ventral part of the ribs. The highest levels of MMP-1 transcripts and MMP-1 protein were found in the metaphyses and diaphyses of the long bones. MMP-1 was expressed by hypertrophic chondrocytes and by osteoblastic cells localized along the newly formed bone trabeculae. No expression was detected in osteoclasts. Two other related members of the MMP family, stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10), were not expressed during days 7.5 and 16.5 of mouse embryogenesis. The tissue-specific expression of MMP-1 and the exclusive ability of interstitial collagenase to digest native collagen of types I, II, III, and X, the major components of bone, cartilage, and tendon, strongly suggests an important and specific function of this enzyme in bone development and remodeling.