Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.     

Clastogenic effect of pesticides in peripheral lymphocytes of cotton-field workers.

We studied clastogenic effects in peripheral lymphocytes of cotton-field workers who were exposed to different pesticides. All the cells were grown in RPMI 1640 medium for 48 and 72 h. The type of aberrations observed in the exposed group are gaps, breaks, dicentrics, exchanges, rings and polyploidy. The frequency of total chromosomal aberrations increased significantly in male pesticide applicators when compared to controls. A significant decrease in mitotic index was observed in the exposed group as compared to the control group. The 48-h cultures showed high incidence of chromosomal aberrations and low mitotic index when compared to 72-h cultures. The difference in chromosomal aberrations between 48- and 72-h cultures was not significant. 24 out of 26 individuals showed ill health effects such as severe giddiness and nervous disorders.     

Metabolism of glomerular basement membrane in short- and long-term streptozotocin diabetic rats

The synthesis of glomerular basement membrane (GBM) total protein and collagen was assessed by two methods in vivo in normal and streptozotocin diabetic rats 4-6 weeks and 42-44 weeks after onset of hyperglycaemia, using L-[2, 3, 3H] proline as a radioactive precursor. The incorporation of tritiated proline into GBM hydroxyproline was used as a measure of collagen synthesis and that into proline as total protein synthesis. The basement membrane fractions from both short- and long-term diabetic rats attained much higher proline and hydroxyproline specific activities compared to normal GBM proline and hydroxyproline specific activities. Early insulin therapy with normalization of blood sugar levels in short-term (4-6 weeks) diabetic rats returned the abnormal increases in GBM total protein and collagen synthesis to normal. By contrast, poor glycaemic control with insulin did not prevent the increases in GBM protein synthesis. The results of the present study suggest that overall enhancement of GBM protein synthesis occurs in both short- and long-term streptozotocin diabetes. Early insulin therapy with normalization of blood sugar levels prevents this increase in GBM protein synthesis. Poor glycaemic control had no effect on abnormal GBM protein synthesis. This may be of potential significance in view of preventing chronic diabetic microvascular complications such as nephropathy.     

Differentiation of canalicular cell processes in bone cells by basement membrane matrix components: regulation by discrete domains of laminin

We have investigated the interaction of rat primary calvarial bone cells and a mouse osteoblast-like cell line MC3T3-E1 with basement membrane components. On a reconstituted gel of basement membrane, both cell types attached and formed isolated clusters that developed long interconnecting cell processes similar to the canalicular network observed in bone. The differentiation of the osteoblastic phenotype was stimulated as determined by increased alkaline phosphatase production and the deposition of mineral. Antibodies to laminin and to a 32/67 kd laminin receptor blocked this differentiation. Cell morphology was altered by the addition of active laminin-derived synthetic peptides, YIGSR-NH2 and CSRARKQAASIKVAVSADR-NH2, but not by an active RGD-containing peptide. When coated directly on plastic, all three peptides promoted cell adhesion, demonstrating that bone cells interact with specific molecular domains of laminin. These data demonstrate that basement membrane plays a key role in formation of a network of cytoplasmic processes resembling the osteocyte canalicular network in bone.     

Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: implications for development

The interaction of transforming growth factor beta (TGF beta) with extracellular matrix macromolecules was examined by using radiolabeled TGF beta and various matrix macromolecules immobilized on nitrocellulose. TGF beta bound to collagen IV with greater affinity than to other extracellular matrix macromolecules tested. Neither laminin nor fibronectin, both of which bind type IV collagen, interfered with the binding of TGF beta to type IV collagen. TGF beta 2 competed effectively with TGF beta 1 for binding to type IV collagen. The biological effect of TGF beta was tested by an assay based on inhibition of proliferation of an osteoblast cell line, MC3T3-E1. The results demonstrated that the effect of TGF beta 1 was sustained when cells were grown on type IV collagen compared to cells grown on laminin, collagen type I, and plastic. These results demonstrate that extracellular matrix components may function as an affinity matrix for binding and immobilizing soluble growth and differentiation factors. In view of the demonstrated role of basement membranes in development the present results imply an important function for transforming growth factor beta bound to collagen IV in local regulation of cell proliferation and differentiation.     

Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.

Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development.     

Transforming growth factor-beta: Its effect on phenotype reexpression by dedifferentiated chondrocytes in the presence and absence of osteogenin

Summary The single and combined actions of transforming growth factor (TGF)-beta and osteogenin were evaluated with regard to induction of colony formation and reexpression of the differentiated phenotype by dedifferentiated rabbit articular chondrocytes in soft agarose under serum-free conditions. TGF-beta alone did not promote colony formation and induced accumulation of proteoglycans and type II collagen at significantly lower levels than those induced by osteogenin. Although synergism between these two growth factors occurred with respect to the induction of colony formation, their joint action on reexpression of the differentiated phenotype was additive. Complex interactions between the two growth factors may explain the latter phenomenon.     

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.     

Effect of pregnancy on serum alanine concentration in normal and genetically diabetic mice.

Serum alanine concentration was determined in nonpregnant and pregnant normal control Swiss albino (SA) and genetically diabetic KK mice. The serum alanine levels were significantly lower in nonpregnant KK than in nonpregnant SA mice. Fasting elicited hypoglycemia, hypoinsulinemia and hypoalaninemia in both groups of pregnant mice. Oral administration of alanine in nonpregnant and pregnant mice resulted in a significant rise in blood sugar levels within 15 min in both groups. However, the initial blood sugar response to oral alanine was greater in pregnant than in nonpregnant mice. This increase in blood sugar response to exogenous alanine appears to be mediated by glucagon. The data suggest that pregnancy elicits hypoglycemia, hypoinsulinemia and hypoalaninemia in both nondiabetic and diabetic mice.     

Dissociative extraction and partial purification of osteogenin, a bone inductive protein, from rat tooth matrix by heparin affinity chromatography

Implantation of demineralized tooth matrix in subcutaneous sites results in new bone formation locally. The osteoinductive activity of the tooth matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the residue was devoid of biologic activity. The bone inductive protein, osteogenin, was partially purified by heparin affinity chromatography. The heparin binding fraction initiated the bone differentiation cascade when implanted with guanidine extracted, inactive bone or tooth matrices. These results imply a cooperative interaction between the soluble osteogenin and collagenous substratum in bone induction.