Solanum muricatum Ameliorates the Symptoms of Osteogenesis Imperfecta In Vivo.

Pepino (Solanum muricatum), which is an evergreen plant native to South America, is well-known for its effects in antioxidation, antidiabetic activity, anti-inflammation, and antitumor activity. A previous study in our lab indicated that Solanum muricatum (SM) extract promoted osteogenic differentiation by upregulating Wnt and BMP signaling pathway in rat bone marrow stromal cells. The osteogenesis imperfecta (OI) mouse model was used in order to further discover the osteogenic properties of SM extract in the present research. We utilized microCT analysis to collect bone mass and microarchitectural parameters at vertebrae and at femur metaphysis in OI mice. Raman spectrometry was applied to identify change of bone mineral and matrix composition during SM treatment. Finally, collagen synthesis marker PINP and collagen degradation marker CTX were detected using enzyme immunoassay. SM extract could improve the bone mass and microarchitectural parameters both at vertebrae and at femur metaphysis. It also significantly increased the collagen content by promoting its biosynthesis and inhibiting its degradation. By using heterozygous Col1a1Jrt /+ mice as a model of OI, 6 weeks treatment of SM extract could significantly ameliorate the symptoms in OI mice. Thus, SM holds potential for developing new drugs of bone formation and bone remodeling.

Osteoblast ontogeny and implications for bone pathology: an overview.

Osteoblasts are specialized mesenchyme-derived cells accountable for bone synthesis, remodelling and healing. Differentiation of osteoblasts from mesenchymal stem cells (MSC) towards osteocytes is a multi-step process strictly controlled by various genes, transcription factors and signalling proteins. The aim of this review is to provide an update on the nature of bone-forming osteoblastic cells, highlighting recent data on MSC-osteoblast-osteocyte transformation from a molecular perspective and to discuss osteoblast malfunctions in various bone diseases. We present here the consecutive stages occurring in the differentiation of osteoblasts from MSC, the transcription factors involved and the role of miRNAs in the process. Recent data concerning the pathogenic mechanisms underlying the loss of bone mass and architecture caused by malfunctions in the synthetic activity and metabolism of osteoblasts in osteoporosis, osteogenesis imperfecta, osteoarthritis and rheumatoid arthritis are discussed. The newly acquired knowledge of the ontogeny of osteoblasts will assist in unravelling the abnormalities taking place during their differentiation and will facilitate the prevention and/or treatment of bone diseases by therapy directed against altered molecules and mechanisms.

Pregnancy- and lactation-associated transient osteoporosis of both hips in a 32 year old patient with osteogenesis imperfecta.

Combination of osteogenesis imperfecta (OI), pregnancy, and transient osteoporosis (TO) of the hip is rare, only a few cases have been published so far. We report a 32 year old woman with OI, with TO on the right hip in her late third trimester. Non-pharmacological measures such as non-weight-bearing resulted in complete remission. Shortly after weaning, TO of the contralateral hip developed and non-pharmacological measures remained ineffective this time. Under treatment with a prostaglandin I(2) analog (iloprost), i.v. bisphosphonate (pamidronate), calcium and vitamin D supplementation rapid improvement of pain and complete remission was achieved.

Benefits of pamidronate in children with osteogenesis imperfecta: an open prospective study.

OBJECTIVES: To study the efficacy of pamidronate in children with osteogenesis imperfecta (OI). PATIENTS AND METHODS: Twenty-nine patients (median age 8.7 years), were given pamidronate in cyclic infusions of 3 days. Patients received 3-13 cycles (median 6), at a dose of 0.5 mg/kg/day in infants (below 2 years of age) and 1 mg/kg/day in children (2 years and older). The interval time between cycles was 2 months in infants and 4 months in children. The median follow-up was 16 months. All patients received daily supplementation of calcium, vitamin D and physical rehabilitation. Assessments were performed at baseline and before each cycle. Fracture rate under treatment was compared to the one in the pre-treatment period. RESULTS: Pain decreased after the first infusion cycle (P < 0.0001). The median of fracture incidence decreased from 15 to 0.5 per year in infants and from 2.0 to 1 per year in children (P = 0.04). Alkaline phosphatase decreased by 31.2% and N-telopeptide collagen cross-links decreased by 61.8% (P < 0.001). Bone mineral density (BMD) of the spine increased by a median of 55.4% (P < 0.001). Z-scores increased from a median of -4.7 to -2.6 (P < 0.001). The femoral neck, BMD increased by a median of 16%. The area of the first four lumbar vertebrae increased by a median of 21.5% (P < 0.001). No adverse effect on growth or on fracture healing was observed. Side effects were symptomatic hypocalcemia in one infant, and the transient acute phase reaction. CONCLUSION: Pamidronate increases BMD, decreases bone remodeling markers, pain and fracture rate in infants and children with OI.

Correction of a mineralization defect by overexpression of a wild-type cDNA for COL1A1 in marrow stromal cells (MSCs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects.

Gene therapy for dominant-negative disorders presents a more difficult challenge than gene therapy for recessive disorders, since even partial replacement of a protein for a recessive disorder can reverse symptoms. Osteogenesis imperfecta (OI) has frequently served as a model disorder for dominant-negative defects of structural proteins. The disease is caused by mutations in type I collagen (COL1A1), the major structural component of bone, skin and other connective tissues. The severity of the phenotype is largely dependent on the ratio of normal to mutant type I procollagen synthesized by cells. Recently, attempts have been made to develop strategies for cell and gene therapies using the adult stem cells from bone marrow referred to as mesenchymal stem cells or marrow stromal cells (MSCs). In this study, we used MSCs from a patient with type III OI who was heterozygous for an IVS 41A+4C mutation in COL1A1. A hybrid genomic / cDNA construct of COL1A1 was transfected into the MSCs and the transfectants were expanded over a 200-fold. Transfected MSCs showed increased expression of the wild-type mRNA and protein. In vitro assays demonstrated that the transfected cells more efficiently differentiated into mineralizing cells. The results indicated that it is possible to overexpress COL1A1 cDNA in OI MSCs and thereby to correct partially the dominant-negative protein defect.

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta.

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.

Abnormal mineral composition of osteogenesis imperfecta bone as determined by electron probe X-ray microanalysis on conventional and cryosections.

Osteogenesis imperfecta (OI) is a genetic disorder of the connective tissue characterized by frequent bone fractures. The cause of bone fragility is still unknown even though substantial work on collagen has been done. We measured the calcium to phosphorus ratio (Ca/P) of bone mineral from 35 OI bone samples and 25 age- and site-matched control specimens, using electron probe X-ray microanalysis in the transmission electron microscope. Ultra-thin cryosections and conventionally prepared resin sections were used. Cryo-ultramicrotomy avoids any possible artifactual demineralization that may occur in conventional aqueous media. The Ca/P ratio obtained by these two methods was compared and there was no statistical difference between them. The results were differentiated according to the clinical types of OI for the first time. The Ca/P ratio of OI bone mineral was lower than normal in both resin and cryosections, and mirrored the severity of the disease. OI type II had the lowest ratio (Ca/P = 1.49) compared with normal age- and site-matched controls (Ca/P = 1.69). This abnormal mineral composition in OI type II could be a contributory factor to bone fragility in OI bone.

Conducta anestésica en una paciente obstétrica con osteogénesis imperfecta / Anesthetic conduct in a obstetric patient with osteogenesis imperfecta

Se reporta en este artículo el caso de una paciente con Osteogénesis Imperfecta (OI), sometida a cesárea abdominal electiva. Esta rara enfermedad genética se caracteriza por una alteración del tejido conectivo que afecta huesos, escleróticas y oído interno, entre otras estructuras. En función de la programación de la cirugía, del carácter obstétrico de la misma y de las recomendaciones bibliográficas consultadas respecto del manejo anestésico de esta infrecuente patología, se decidió realizar una técnica regional subaracnoidea. El procedimiento quirúrgico y el curso anestésico no presentaron complicaciones. Por otra parte, se citan otros casos reportados en la literatura médica. Se analiza la conducta anestésica a seguir en cuanto a la evaluación preanestésica, el monitoreo intraoperatorio, las medidas generales de protección y la elección de la técnica anestésica adecuada con sus fundamentos. Finalmente, se concluye que la anestesia subaracnoidea, por su menor dificultad técnica y el uso de menor dosis de anestésico local, resulta ser una elección segura y racional en una paciente obstétrica con Osteogénesis Imperfecta.

Conducta anestésica en una paciente obstétrica con osteogénesis imperfecta / Anesthetic conduct in a obstetric patient with osteogenesis imperfecta

Se reporta en este artículo el caso de una paciente con Osteogénesis Imperfecta (OI), sometida a cesárea abdominal electiva. Esta rara enfermedad genética se caracteriza por una alteración del tejido conectivo que afecta huesos, escleróticas y oído interno, entre otras estructuras. En función de la programación de la cirugía, del carácter obstétrico de la misma y de las recomendaciones bibliográficas consultadas respecto del manejo anestésico de esta infrecuente patología, se decidió realizar una técnica regional subaracnoidea. El procedimiento quirúrgico y el curso anestésico no presentaron complicaciones. Por otra parte, se citan otros casos reportados en la literatura médica. Se analiza la conducta anestésica a seguir en cuanto a la evaluación preanestésica, el monitoreo intraoperatorio, las medidas generales de protección y la elección de la técnica anestésica adecuada con sus fundamentos. Finalmente, se concluye que la anestesia subaracnoidea, por su menor dificultad técnica y el uso de menor dosis de anestésico local, resulta ser una elección segura y racional en una paciente obstétrica con Osteogénesis Imperfecta. (AU)

Three novel type I collagen mutations in osteogenesis imperfecta type IV probands are associated with discrepancies between electrophoretic migration of osteoblast and fibroblast collagen.

In three cases of type IV osteogenesis imperfecta (OI), we identified unique point mutations in type I collagen alpha1(I) cDNA. In two cases, the appearance of dimers indicated the presence of cysteine substitutions in the alpha1(I) protein chain. Cyanogen bromide digestion localized these cross-links to CB8 and 3, respectively. In the third case, the overmodification pattern of the CNBr peptides was compatible with a substitution in the aa 123-402 region of either type I collagen chain. We identified a unique point mutation in each proband, which resulted in substitutions for glycine residues in a 300-aa region of the alpha1(I) helix, specifically, Gly to Ala at codon 220 (GGT-->GCT), Gly to Cys at codon 349 (GGT-->TGT) and Gly to Cys at codon 523 (GGT-->TGT). We compared each proband's fibroblast and osteoblast collagen directly, as well as with fibroblast and osteoblast controls. For all cases, the OI osteoblast collagen was more electrophoretically delayed than OI fibroblast collagen. In the patient with G349C, OI fibroblast and osteoblast collagen synthesized in the presence of alpha,alpha'-dipyridyl co-migrated on gels, demonstrating that the electrophoretic discrepancy resulted from differences in post-translational modification. Melting temperature curves for stability of the collagen helix yielded an identical Tm for control fibroblast and osteoblast collagen (41.2 degrees C). By contrast, for collagen with the gly349-->cys substitution, the Tm of the fibroblast collagen was 1 degree C lower than the Tm of the osteoblast collagen. These data indicate that the metabolism of mutant collagen might be cell-specific and has significant implications for understanding the phenotype/genotype correlations and the pathophysiology of OI.

Long-term extracellular matrix metabolism by cultured human osteogenesis imperfecta osteoblasts.

Osteopenia due to deficient extracellular matrix synthesis is a hallmark of osteogenesis imperfecta (OI), Previous studies carried out within 72 h of osteoblast subculture, at an early stage of matrix synthesis, indicated that for osteoblasts derived from human OI patients the total amounts of collagen, osteonectin, and three proteoglycans were significantly reduced, while total amounts of thrombospondin, fibronectin, and matrix hyaluronan were elevated compared with age-matched controls. The current study was undertaken to follow OI osteoblast matrix metabolism as that matrix is synthesized, deposited, and matured. Steady-state metabolic radiolabeling was used to follow the metabolism of collagen, hyaluronan, and total proteoglycan by OI and normal osteoblasts for up to 5 weeks. Trabecular osteoblasts from non-OI controls showed an increase in total and matrix-associated collagen synthesis during the first and second week, reaching steady-state levels by week 4. In contrast, cultured OI osteoblasts did not increase either the total (medium + matrix-associated) or matrix-associated collagen during the entire 5-week period. Proteoglycan synthesis exhibited a pattern similar to that for collagen. OI-derived proteoglycans differed from controls in that levels in OI cultures did not reflect the normal time-dependent increase in total proteoglycan and proteoglycan matrix deposition. OI osteoblast hyaluronan synthesis was increased when compared with age-matched controls during 4 weeks of culture. In contrast, the ratios of calcium to phosphorus solublized from control and the OI extracellular matrix were not statistically different. Thus, with respect to the synthesis of collagen, proteoglycans, and hyaluronan, OI osteoblasts fail to parallel controls in depositing and elaborating extracellular matrix during 35 days in culture.

An electron probe X-ray microanalytical study of bone mineral in osteogenesis imperfecta.

A semiquantitative electron probe X-ray microanalytical (XRMA) technique, in conjunction with transmission electron microscopy, was used to compare the calcium to phosphorus (Ca/P) molar ratios in calcium phosphate standards of known composition, in normal bone and in bone from patients with osteogenesis imperfecta (OI). Using a modified routine processing and resin embedding schedule, the measured Ca/P molar ratio of calcium phosphates standards of known composition were found to correlate well with the Ca/P molar ratio based on their respective chemical formulae. This technique was then used to compare the Ca/P molar ratio in normal human bone and in OI bone. The Ca/P ratio values for normal bone (Ca/P = 1.631) correlated well with those for chemically prepared hydroxyapatite (Ca/P = 1.602), but in bone from OI patients, the Ca/P molar ratio was significantly lower (Ca/P = 1.488). This study has shown that there is a lower Ca/P molar ratio in OI bone compared with normal, matched bone. This suggests that the mineral deviates from the carbanoapatite usually found in bone. Isomorphous substitutions in the carbanoapatite lattice could account for this although this study has neither proved nor disproved this. The altered bone mineral is another factor that could contribute to the increased fracture rate observed in OI.

Chemical abnormalities.

This article provides an in-depth summary of standard and recently developed biochemical assays that are useful for the evaluation of the patient with metabolic bone disease. In addition, each of the common metabolic bone diseases is discussed in the context of the associated chemical abnormalities.

Fluoride therapy in metabolic bone disease.

Multiple forms of treatment are used for osteoporosis, Paget's disease of the bone, and osteogenesis imperfecta. Because of the effect of fluoride (F) on bone, these bone conditions may benefit from F therapy. In the present study, the metabolic effects of two dose levels of F (20 and 45 mg, given as sodium fluoride) were studied on calcium (Ca) and F metabolism in patients with osteoporosis, whereas the effect of the 45-mg dose was studied in patients with Paget's disease. In patients with osteoporosis, the retention of F increased with increasing F intake, indicating F deposition in bone. However, only the 45-mg dose decreased urinary Ca excretion. F did not decrease fecal Ca excretion, indicating that the intestinal absorption of Ca did not improve during F therapy. This was confirmed in 47Ca absorption studies. The duration of F therapy was 3 months, utilizing the 45-mg dose. Clinically, the patients experienced relief of bone pain, and the onset of this change coincided with the decrease in urinary Ca. The 3-month course of treatment was sufficient for alleviation of clinical symptoms for many months and even years. In patients with Paget's disease, F supplements decreased urinary Ca and also resulted in relief of bone pain. F therapy for osteogenesis imperfecta resulted in decreased fracture incidence.

Vitamin D metabolism in osteogenesis imperfecta during calcitonin therapy.

The effect of calcitonin on plasma concentrations of 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] was studied in six patients with osteogenesis imperfecta. The mean pretreatment value of plasma 1,25-(OH)2D was significantly higher than the mean value of age-matched control subjects (P less than .05). High or normal plasma levels of 1,25-(OH)2D before calcitonin therapy were decreased after 1 month of therapy and remained normal thereafter in all six patients. Plasma 25-hydroxyvitamin D concentrations, which were normal before calcitonin injection, remained normal during calcitonin administration. These results indicate that there may be acute and chronic effects of calcitonin on vitamin D metabolism.