Stimulation of systemic bone formation induced by experimental blood loss.

Direct physical injury to bone marrow is associated with a systemic osteogenic response. However, blood loss, a condition that stimulates hemopoietic stem cells, also may activate osteoprogenitor cells in the bone marrow. To determine if bleeding induces a systemic osteogenic response, the mineral appositional rates and osteoblast numbers were determined in the bones of rats that were subjected to controlled cardiac bleeding and compared with those of rats subjected to ablation of their tibial bone marrow. In addition, a study of the kinetics of the osteogenic responses during the first 10 days after operative treatment was performed by quantitating the serum levels of biochemical indices known to be associated with systemic bone formation. The results showed that animals that sustained acute blood loss (1% or 3% body weight) or injury to their tibial bone marrow had statistically significant increases in mineral appositional rate, osteoblast number, and serum levels of osteogenic growth peptide. The kinetics studies showed that osteogenic growth peptide levels peaked on the tenth postoperative day and declined sharply thereafter. An enhancement of serum osteocalcin activity occurred only on the second postoperative day, was increased in all experimental groups when compared with untreated control animals, but immediately declined to baseline levels. Alkaline phosphatase activities increased in the experimental groups, peaking on Day 10 after tibial bone marrow ablation and on Day 12 in the group that underwent bleeding. These findings suggest that bleeding alone, independent of any skeletal trauma, may evoke a systemic osteogenic response. This response is similar in its timing and magnitude to that which has been shown to follow direct physical injury to bone marrow. The observation that systemic bone formation follows bone marrow activation induced by two different stimuli suggests that these responses may be mediated by common regulatory mechanisms. The ability to trigger or control these responses may form the basis for future therapeutic strategies to enhance bone formation.

Postablation bone marrow regeneration: an in vivo model to study differential regulation of bone formation and resorption.

The postablation bone marrow regeneration model is an in vivo paradigm of synchronous bone formation and resorption restricted to a defined reference anatomical location. The blood clot that fills the medullary cavity immediately after marrow removal is organized by replacement with primary cancellous bone. At the peak of the osteogenic phase almost the entire medullary cavity is filled with the trabecular mesh. The primary bone trabeculae are then subjected to osteoclastic resorption and replacement by intact marrow. Since in animals of defined strain, sex and age the timing and extent of formation and resorption are highly reproducible, the postablation model in combination with simple vital methods and/or bone histomorphometry as well as molecular and biochemical approaches applied at specified time periods provides an efficient in vivo tool to assess the efficacy of antiresorptive agents as well as their possible adverse effects on bone formation. When applied to transgenic animals this model may become useful to determine the role of individual genes in matrix formation, mineralization and resorption.

Regulatory role of osteogenic growth peptide in proliferation, osteogenesis, and hemopoiesis.

Bone marrow regeneration after injury is preceded by local and systemic osteogenic reactions. Recently, an osteogenic growth peptide was characterized in the regenerating marrow. The osteogenic growth peptide also is abundant in normal serum where it is markedly and transiently increased after marrow injury. This increase and the osteogenic growth peptide-induced stimulation of bone formation in vivo suggest a role for this peptide in mediating the systemic osteogenic response. In vitro, the osteogenic growth peptide is an autocrine mitogen for osteoblastic and fibroblastic cells. It also stimulates alkaline phosphatase activity and matrix mineralization. The serum osteogenic growth peptide is downregulated in osteoporotic ovariectomized mice. The osteogenic growth peptide levels as well as the bone loss, tetracycline uptake, and serum osteocalcin are reversed by exogenously administered osteogenic growth peptide. In normal mice, the osteogenic growth peptide increases white blood cell counts and total femoral bone marrow cellularity. These increases include all the hemopoietic lineages. When given to mice for 1 week before ablative radiotherapy and bone marrow transplantation, synthetic osteogenic growth peptide stimulates the bone marrow transplant engraftment; optimal osteogenic growth peptide doses doubled the survival rate. These data indicate that osteogenic growth peptide has an important role in the pathogenesis and treatment of systemic bone loss and provide a basis for further development of an antiosteoporotic osteogenic growth peptide therapy. It is suggested also that the osteogenic growth peptide promotes hemopoiesis secondary to the stimulation of the stromal (particularly osseous) microenvironment.

Polypeptide factors regulating osteogenesis and bone marrow repair.

Osteogenic growth polypeptides regulate bone cell function in vitro and may act in vivo in an autocrine, paracrine, or endocrine manner. Several of these polypeptides are present in the blood in an inactive form. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then, the same and/or other polypeptides expressed by stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This may be an indirect influence since these polypeptides can regulate the stromal cell expression of some of the hemopoietic factors. Clinically, the osteogenic growth polypeptides that regulate osteogenesis and hemopoiesis have a potential role in osteoporosis therapy, implant bone surgery, and bone marrow transplantation.

Regulatory role of osteogenic growth polypeptides in bone formation and hemopoiesis.

Osteogenic growth polypeptides such as the osteogenic growth peptide (OGP), fragments of the parathyroid hormone (PTH), and insulin-like growth factors (IGF) regulate bone cell activity in vitro and may affect in vivo osteoblastic functions in an autocrine, paracrine, or endocrine manner. Several growth polypeptides capable of regulating osteogenesis circulate in the blood in an inactive form, complexed to parent molecules or binding proteins. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then osteogenic growth polypeptides expressed by osteoblasts and other stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This is probably an indirect effect inasmuch as these polypeptides can regulate the stromal cell expression of hemopoietic factors such as macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and the stem cell factor (SCF). The postablation marrow regeneration model is suitable for studying the expression and activity of osteogenic growth polypeptides and already has been used to assess the effect of aging on these parameters. Clinically, the osteogenic growth polypeptides and marrow regeneration have a potential role in osteoporosis therapy, implant and corrective bone surgery, and bone marrow transplantation.

Osteomalacia in hereditary hypophosphatemic rickets with hypercalciuria: a correlative clinical-histomorphometric study.

We characterized the bone disease of transilial biopsy specimens from children with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and genetically related asymptomatic hypercalciuric subjects. All HHRH patients showed irregular mineralization fronts, markedly elevated osteoid surface and seam width, increased number of osteoid lamellae, and prolonged mineralization lag time. These findings are consistent with a mineralization defect and indicate unambiguously that the bone disease in HHRH is osteomalacia. The only abnormality seen in the asymptomatic hypercalciuric subjects was slightly extended osteoid surface. Parametric and nonparametric statistical analyses performed on a pooled sample of HHRH patients and asymptomatic hypercalciuric subjects revealed a very high inverse correlation and a tight linear relationship between serum phosphorus and osteoid parameters. Serum 1,25-dihydroxyvitamin D, which is low in other forms of hereditary hypophosphatemia and osteomalacia, is elevated in HHRH and correlated positively with osteoid parameters and the mineralization lag time. Serum alkaline phosphatase showed similar relationships. These results as well as the clinical, biochemical, and radiological remission of bone disease consequent to phosphate therapy strongly suggest that in HHRH 1) hypophosphatemia alone is sufficient to cause osteomalacia; and 2) the elevation of 1,25-dihydroxyvitamin D reflects the degree of the primary renal phosphate leak, but is not involved in the pathogenesis of the bone disease.

Hormone-responsive cells derived from human dental papilla: characterization in vitro and in vivo in diffusion chambers.

Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E2 there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.

Neutral peptidase activities in matrix vesicles from bovine fetal alveolar bone and dog osteosarcoma.

Extracellular matrix vesicles from bovine fetal alveolar bone and from a dog osteosarcoma were isolated by differential centrifugation and then fractionated on a discontinuous sucrose density gradient. The fractions were examined by electron microscopy and were analyzed for protein, alkaline phosphatase, aminotripeptidase, and four different beta-naphthylamidase activities. The low-density peak of enzyme activities was shown by electron microscopy to be much more homogeneous than the crude matrix vesicle fraction. Two major peaks of protein and enzyme activities were present, one in the high and one in the low density layers. There was good correlation between the activities of alkaline phosphatase and the various peptidases in the fractions from the sucrose density gradient. These results indicate a coexistence of peptidase and alkaline phosphatase in matrix vesicles. On the other hand, there was generally no correlation between the peptidase and alkaline phosphatase activities in vesicular specimens from bovine liver obtained in the same way. Most of the peptidase activity and about half of the alkaline phosphatase activity were solubilized from bone matrix vesicles by detergents. The extracted alkaline phosphatase and alanyl beta-naphthylamidase activities were separated from each other on a DEAE-cellulose column.

Occurrence of actin-like protein in extracellular matrix vesicles.

Preliminary indications of the occurrence of actin and myosin in crude matrix vesicle preparations have been reported previously. In the present study extracellular matrix vesicles from rat alveolar bone were isolated. They were further purified by a sucrose density gradient. SDS-polyacrylamide gel electrophoresis of the purified vesicles revealed the presence of a polypeptide with a molecular weight of 43 K daltons and with electrophoretic mobility identical to that of blood platelet actin. The limited proteolysis of both 43 K dalton vesicular polypeptide and actin by Staphylococcus aureus-V8-protease revealed three fragments with identical electrophoretic mobility. In addition, the vesicular preparations inhibited the activity of DNase I, a property typical of actin monomers. Filamentous material extracted from matrix vesicles showed ultrastructuraL features of F-actin. Reaction of this material with heavy meromyosin resulted in arrowhead formation, which is characteristic of acto-heavy meromyosin. The occurrence of actin in extracellular matrix vesicles may account for their budding from the osteoblastic plasma membrane, their possible motility in the matrix, and maintenance of the spherical shape.

Extracellular matrix vessels in human osteogenic neoplasms: an ultrastructural and enzymatic study.

The results of a study of the ultrastructural and enzymatic features of extracellular matrix vesicles in human osteogenic neoplasms are reported. Specimens from three osteosarcomas, a chondrosarcoma, and an osteoblastoma were processed for electron microscopic study and for preparation of vesicular, membrane, and cellular fractions. Electron micrographs of each lesion showed primary mineralization comprised of matrix vesicles and calcifying nodules. There was a distinct pattern of distribution of enzymatic activity among fractions from the osteosarcomas; namely that the highest values for specific activity of alkaline and pyrosphosphatases and adenosine triphosphases (ATPases) in the vesicle fractions and lowest in the cell fractions. This pattern was not consistent in fractions from the other neoplasms. The aforementioned enzymes are considered essential for the onset of mineralization. The data presented establish the role of matrix vesicles in neoplastic calcification and suggest the need for further studies into the diagnostic value of the vesicles.

A comparative study on the occurrence and activity of extracellular matrix vesicles in young and adult rat maxillary bone.

The occurrence of matrix vesicles in the maxillary bone of normal young and adult rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed that the vesicles are invested in trilaminar membranes. Occasionally, crystals were found both within the vesicles and in the surrounding matrix. Separated fractions of vesicles, membranes, and cells were studied biochemically. The amounts of vesicular protein and enzymatic activity per gram bone in the adult rats were significantly lower than in the young. This coincides with the higher number of vesicles observed in the TEM specimens of young rats when compared to that in the old ones. The specific activities of all enzymes examined in the three bone fractions obtained from both young and adult rats were similar. This indicates that no significant difference exists in the enzymatic characteristics of matrix vesicles from the maxillary bone of young and adult rats.

The occurrence of hydroxyapatite crystals in extracellular matrix vesicles after surgical manipulation of the rabbit knee joint.

Free autologous grafts of synovial tissue were transplanted into experimental defects produced in the articular cartilage of rabbit knee joints. The grafted tissue underwent transformation into fibrocartilage. Extracellular matrix vesicles associated with calcified areas were present at the grafted sites. Hydroxyapatite crystals were found within these vesicles and in their vicinity. No calcification occurred in articular cartilage from sham operated joints in which defects were produced but no grafts made and in normal controls. These tissues showed abundant matrix vesicles devoid of crystalline mineral. A careful study of normal synovial tissue did not reveal matrix vesicles and calcifications. The present observations suggest that matrix vesicles in normal articular cartilage exist in a latent form. Vesicle mineralization following surgical manipulations of the joint is probably a manifestation of the metabolic stage of the tissue.

Simultaneously occurring salivary gland tumors of different types.

Two cases of simultaneously occurring salivary gland tumors of the parotid gland are reported. Both patients were women in the sixth decade of life. In the first patient, a carcinoma in pleomorphic adenoma and an oncocytoma were found in the right parotid gland. The second patient had an adenoid cystic carcinoma associated with a sebaceous cell adenoma. The addition of these cases to the previously published data brings the total number of simultaneously occurring salivary gland tumors of different types to ten.

Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats.

The occurrence of vesicles in the extracellular matrix of alveolar bone of normal young rats was demonstrated by both ultrastructural and enzymatic studies. Transmission electron microscopy revealed abundant vesicles in the matrix. The presence of hydroxyapatite crystals, both within the vesicles and in the matrix, was affiliated with rupture of the vesicular membrane. Calcifying nodules were scarce. High levels of both specific and total activities of alkaline- and pyrophosphatases were found in the fraction of isolated vesicles. This fraction also showed activities of different ATPases and acid phosphatase.

Inflammatory lesions and bone resorption induced in the rat periodontium by lipoteichoic acid of Streptococcus mutans.

Severe inflammatory lesions were induced in the periodontal tissues of the rat following the intragingival injection of lipoteichoic acid (LTA) from Streptococcus mutans. There was no difference in the severity and distribution of the lesions between nonimmunized rats and animals immunized against LTA after antigenic challenge. The lesions are characterized by the occurrence of granulation tissue, massive infiltration of PMNs, abscess formation, bone resorption, and new bone formation. Deacylated LTA and saline caused relatively mild inflammation, and no significant bone resorption or new bone formation was evident. The peak response was reached after 3 intragingival infections. The mechanisms by which LTA caused the pathological alterations in the rat periodontium and the possible relations of this experimental model to periodontal disease in the human are discussed.

The relationship between extracellular matrix vesicles and calcospherities in primary mineralization of neoplastic bone tissue. TEM and SEM studies on osteosarcoma.

Primary mineralization in neoplastic tissue was studied in osteosarcoma, correlating observations obtained by SEM to those found with TEM. The process is characterized by extracellular matrix vesicles, distributed in the matrix between the forming neoplastic cells and the calcifying fronts. The occurrence of osmiophilic material and solitary hydroxyapatite crystals within the vesicles is followed by accumulation of apatite crystals, disappearance of the vesicular membrane and formation of calcospherites and calcified fronts. The process described here in neoplastic tissue is essentially similar to primary calcification in normal calcified tissues.

Correlative transmission and scanning electron microscopy of the initial mineralization of healing alveolar bone in rats.

Primary mineralization in healing sockets after extraction of molar teeth was studied in rats. The observations obtained by scanning electron microscopy were correlated by transmission electron microscopy. The process is characterized by abundance of extracellular matrix vesicles distributed between the forming cells and the calcifying fronts. The occurrence of osmiophilic material and solitary hydroxyapatitte crystals within the vesicles was followed by accumulation of hydroxyapatite crystals, disappearance of the vesicular membrane and formation of calcospherites that conglomerate into calcified fronts. The process described here in bone healing is essentially similar to primary mineralization in other normal and pathological calcified tissues.

Dermoid cyst of the floor of the mouth in an infant. (Case report).

Dermoid cysts of the floor of the mouth in the infant are rarely encountered although the aetiology is mainly embryological. Meyer proposes the classification of the cysts based on the histological differentiation of the elements of the cyst wall. A case is reported which shows differences in the epithelial lining of the cysts found followed by the differential diagnosis and treatment.