Endocannabinoids and the regulation of bone metabolism.

In mammals, including humans, bone metabolism is manifested as an ongoing modelling/remodelling process whereby the bone mineralised matrix is being continuously renewed. Recently, the main components of the endocannabinoid system have been reported in the skeleton. Osteoblasts, the bone forming cells, and other cells of the osteoblastic lineage, as well as osteoclasts, the bone resorbing cells, and their precursors, synthesise the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG). CB(1) cannabinoid receptors are present in sympathetic nerve terminals in close proximity to osteoblasts. Activation of these CB(1) receptors by elevated bone 2-AG levels communicates brain-to-bone signals as exemplified by traumatic brain injury-induced stimulation of bone formation. In this process, the retrograde CB(1) signalling inhibits noradrenaline release and alleviates the tonic sympathetic restrain of bone formation. CB(2) receptors are expressed by osteoblasts and osteoclasts. Their activation stimulates bone formation and suppresses bone resorption. CB(2)-deficient mice display a markedly accelerated age-related bone loss. Ovariectomy-induced bone loss can be both prevented and rescued by a CB(2) specific agonist. Hence, synthetic CB(2) ligands, which are stable and orally available, provide a basis for developing novel anti-osteoporotic therapies, free of psychotropic effects. The CNR2 gene (encoding CB(2)) in women is associated with low bone mineral density, offering an assay for identifying females at risk of developing osteoporosis.

Cannabinoid receptors and the regulation of bone mass.

A functional endocannabinoid system is present in several mammalian organs and tissues. Recently, endocannabinoids and their receptors have been reported in the skeleton. Osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells, produce the endocannabinoids anandamide and 2-arachidonoylglycerol and express CB2 cannabinoid receptors. Although CB2 has been implicated in pathological processes in the central nervous system and peripheral tissues, the skeleton appears as the main system physiologically regulated by CB2. CB2-deficient mice show a markedly accelerated age-related bone loss and the CNR2 gene (encoding CB2) in women is associated with low bone mineral density. The activation of CB2 attenuates ovariectomy-induced bone loss in mice by restraining bone resorption and enhancing bone formation. Hence synthetic CB2 ligands, which are stable and orally available, provide a basis for developing novel anti-osteoporotic therapies. Activation of CB1 in sympathetic nerve terminals in bone inhibits norepinephrine release, thus balancing the tonic sympathetic restrain of bone formation. Low levels of CB1 were also reported in osteoclasts. CB1-null mice display a skeletal phenotype that is dependent on the mouse strain, gender and specific mutation of the CB1 encoding gene, CNR1.

Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice.

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

Mitogenic G(i) protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: activation by C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)] and attenuation of activation by cAMP.

In osteogenic and other cells the mitogen-activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP-binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C-terminal pentapeptide OGP(10-14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10-14), but not OGP, enhance MAP kinase activity in a time-dependent manner. The OGP(10-14)-induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a G(i) protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide-activated, mitogenic Gi protein-MAP kinase-signaling cascade. Forskolin and dBu(2)-cAMP abrogated the OGP(10-14)-stimulated proliferation, but induced only 50% inhibition of the OGP(10-14)-mediated MAP kinase activation, suggesting additional MAP kinase-dependent, OGP(10-14)-regulated, cellular functions. Finally, it is demonstrated that OGP(10-14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP-OGPBP complexes.

Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)].

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10-14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or D-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with L- or D-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and L- or D-Ala substitution of Gly13 resulted in 70-97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10-14).

Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells.

The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10-14). The OGP(10-14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10-14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP-OGP binding protein (OGPBP) complex formation, sOGP(10-14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10-14) is a naturally occurring human and murine mitogen. In addition, the data suggests that the OGP(10-14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP-OGPBP complexes.

Biosynthesis of osteogenic growth peptide via alternative translational initiation at AUG85 of histone H4 mRNA.

The osteogenic growth peptide (OGP) is an extracellular mitogen identical to the histone H4 (H4) COOH-terminal residues 90-103, which regulates osteogenesis and hematopoiesis. By Northern analysis, OGP mRNA is indistinguishable from H4 mRNA. Indeed, cells transfected with a construct encoding [His102]H4 secreted the corresponding [His13]OGP. These results suggest production of OGP from H4 genes. Cells transfected with H4-chloramphenicol acetyltransferase (CAT) fusion genes expressed both "long" and "short" CAT proteins. The short CAT was retained following an ATG --> TTG mutation of the H4 ATG initiation codon, but not following mutation of the in-frame internal ATG85 codon, which, unlike ATG1, resides within a perfect context for translational initiation. These results suggest that a PreOGP is translated starting at AUG85. The translational initiation at AUG85 could be inhibited by optimizing the nucleotide sequence surrounding ATG1 to maximally support upstream translational initiation, thus implicating leaky ribosomal scanning in usage of the internal AUG. Conversion of the predicted PreOGP to OGP was shown in a cell lysate system using synthetic [His102]H4-(85-103) as substrate. Together, our results demonstrate that H4 gene expression diverges at the translational level into the simultaneous parallel production of both H4, a nuclear structural protein, and OGP, an extracellular regulatory peptide.

Human alpha 2-macroglobulin is an osteogenic growth peptide-binding protein.

The osteogenic growth peptide (OGP) is a 14mer mitogen of osteoblastic and fibroblastic cells. Physiologically, OGP is present in high abundance in human and other mammalian sera. Most of the serum OGP is complexed noncovalently to heat sensitive, high molecular weight OGP-binding proteins (OGPBPs). Changes in serum OGP levels that follow bone marrow ablation and the low doses of exogenous OGP required for the stimulation of bone formation suggest a regulatory role for the OGPBPs. In the present work, the OGP binding activity was monitored by competitive binding to [3-125I(Tyr10)]-sOGP and the corresponding complexes were demonstrated on nondenaturing cathodic polyacrylamide gel electrophoresis. We show that OGP binds to both native and activated human plasma alpha 2-macroglobulin (alpha 2M). alpha 2M was also immunoidentified in reduced and nonreduced SDS-polyacrylamide gel electrophoresis of OGP-affinity purified plasma-derived proteins. Immunoreactive OGP was detected in commercial preparations of both forms of alpha 2M; OGP was purified to homogeneity from the commercial preparation of activated alpha 2M. In MC3T3 E1 cells, native alpha 2M, at concentrations < 50 ng/mL, had a substantially increased mitogenic effect in the presence of synthetic, native-like, OGP (sOGP). Similar amounts of activated alpha 2M inhibited the sOGP proliferative effect. These results suggest that the native alpha 2M enhances the immediate availability of OGP to its target cells. Activated alpha 2M may participate in the removal of OGP from the system.

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.

Isolation of osteogenic growth peptide from osteoblastic MC3T3 E1 cell cultures and demonstration of osteogenic growth peptide binding proteins.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals in increases osteogenesis and hemopoiesis. In stromal cell cultures OGP stimulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP in high abundance is present in normal human and animal serum mainly complexed to OGP binding protein (OGPBP) or proteins. Here we show the presence of two OGPBPs, OGPBP-1, and OGPBP-2, in cultures of osteoblastic MC3T3 E1 cells. Immunoreactive OGP (irOGP) also accumulates in the medium of these cultures and in cultures of NIH 3T3 fibroblasts. A large amount of irOGP was released by heat inactivation of OGPBP-2 and purified by ultrafiltration and hydrophobic HPLC. The purified irOGP was identical to OGP obtained previously from rat regenerating bone marrow and human serum in terms of its amino acid sequence, immunoreactivity, and mitogenicity. Osteoblastic and fibroblastic cell proliferation can be arrested by anti-OGP antibodies and rescued by exogenous OGP, indicating that in the absence of serum or other exogenous growth stimulators the endogenously produced OGP is both necessary and sufficient for baseline proliferation. The OGP production is up- and down-regulated, respectively, by low and high doses of exogenous OGP in a manner consistent with an autoregulated feedback mechanism. The most effective OGP dose in MC3T3 E1 cells is at least two orders of magnitude lower than that in non-osteoblastic cell systems. This differential sensitivity of the osteoblastic cells could result in a preferential anabolic effect of OGP in bone.

Osteogenic growth peptide increases blood and bone marrow cellularity and enhances engraftment of bone marrow transplants in mice.

The osteogenic growth peptide (OGP) was characterized recently in regenerating bone marrow (BM) and normal serum. In vitro, the OGP regulates stromal-cell proliferation and differentiated functions. In vivo, an increase in serum OGP accompanies the osteogenic phase of postablation BM regeneration. The present results in normal mice show that OGP induces a balanced increase in WBC counts and overall BM cellularity. In mice receiving myeloablative irradiation and syngeneic or semiallogeneic BM transplants, OGP stimulates hematopoietic reconstruction and doubles the survival rate; these effects are dependent on initiating the OGP administration before irradiation. Chimerism measurements in semiallogeneic graft recipients suggest no preferential effect of OGP on residual host cells. The data implicate OGP in the acceleration of hematopoiesis secondary to expansion of the stromal microenvironment and/or enhancement of stroma-derived signals to stem cells. The low-dose effectiveness of OGP is explained by the demonstration of an autocrine positive feedback loop that together with the OGP-binding protein sustains high serum levels of the peptide. A potential OGP-based treatment in combination with chemoradiotherapy is attractive because of the OGP-induced balanced multi-lineage enhancement of hematopoiesis and possible replacement of expensive recombinant cytokines by a readily synthesized peptide.

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.

Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals, OGP increases osteogenesis. Immunoreactive OGP (irOGP) in high abundance was demonstrated in normal animal serum mainly as an OGP-OGP-binding protein (OGPBP) complex. Here we show the presence of an OGP-OGPBP system in normal human serum. The total irOGP content, of which the bound peptide comprises at least 80-90%, ranged from 480-4460 mumol/L, several orders of magnitude higher than that of other regulatory polypeptides. The steady state/total irOGP ratio declined between 23 and 49 yr of age. The bound irOGP, purified by boiling, ultrafiltration, and hydrophobic high pressure liquid chromatography, was identical to OGP obtained previously from rat regenerating marrow and mouse stromal cell cultures in terms of its amino acid sequence, immunoreactivity, and mitogenicity. These data demonstrate the usefulness of our immunoassay to measure circulating OGP. More importantly, the identity of the human OGP with that of other species indicates the peptide's evolutionary conservation and, thus, its biological importance. The natural occurrence of OGP in man signifies its potential role in the prevention of bone loss and rescue of bone mass, especially in osteoporosis.

Osteogenic growth peptide regulates proliferation and osteogenic maturation of human and rabbit bone marrow stromal cells.

The recently discovered osteogenic growth peptide (OGP) has been shown to regulate proliferation in fibroblastic and osteoblastic cell lines derived from rats and mice and also alkaline phosphatase activity in the latter was found to be affected. In vivo the OGP enhances bone formation and trabecular bone density. The results of the current study indicate that the OGP is also a potent regulator of marrow stromal cells from man and rabbit, as well as rabbit muscle fibroblasts. The main OGP activity in both marrow systems is a marked stimulation of alkaline phosphatase activity and matrix mineralization. In the rabbit-derived cell culture this enhancement is accompanied by a reciprocal inhibition of proliferation. On the other hand, the human cells show a concomitant increase of both parameters. The proliferative effect of the OGP is similar to that of growth hormone (GH) and basic fibroblast growth factor (bFGF). The combined activity of the OGP with GH is smaller than that of each of the polypeptides alone. The OGP and bFGF potentiate each other. Of the three polypeptides tested, OGP is the most potent enhancer of alkaline phosphatase activity and mineralization. bFGF has no influence on these characteristics of osteogenic maturation. The OGP maturational activity is unaffected by either GH or bFGF. These data suggest that the marrow stromal cells serve as targets for the OGP that mediate the OGP-induced increase in osteogenesis. The effect on the human cells implies a role for the OGP in clinical situations where the osteogenic potential of bone marrow is involved.

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.

Mitogenic action of osteogenic growth peptide (OGP): role of amino and carboxy-terminal regions and charge.

We have recently reported the discovery of a 14-amino-acid osteogenic growth peptide (OGP). In vivo OGP increases bone formation and trabecular bone density. Physiologically it is found in serum complexed to an OGP binding protein (OGPBP). In vitro OGP has a biphasic effect on osteoblastic MC 3T3 E1 and fibroblastic NIH 3T3 cell proliferation; at low concentrations (0.01-1.0 and 1.0-100.0 pM, respectively) it is highly stimulatory with an inhibition at higher doses. To assess possibilities of labeling synthetic OGP to obtain radio- or fluorescent ligands, OGP analogues were extended at the N- or C-termini with Cys or Cys(S-NEtSucc) or the OGP Tyr-10 replaced by 3-I(Tyr). All analogues with N-terminal modifications, as well as the [Cys15]OGP-NH2 retained the OGP-like dose-dependent effect on proliferation of the MC 3T3 E1 and NIH 3T3 cells, although the magnitude of stimulation was lower, approx. 2/3 that of the native-like synthetic OGP. The [Cys15(S-NEtSucc)]OGP-NH2 and [3-I(Tyr10)]OGP shared only the inhibitory activity of OGP. This suppression is further shared by a number of other positively and negatively net charged, but not net neutral, peptides. Both N-terminal-modified analogues displayed a decreased binding activity to the OGPBP. All analogues except reverse OGP, [3-I(Tyr10)]OGP and [Cys15(S-NEtSucc)]OGP-NH2 reacted with anti-OGP antibodies. These data are not only important for labeling purposes but suggest a respective role for the OGP N-and C-terminal regions in binding to the OGPBP and putative OGP receptor. It appears that the OGP proliferative activity represents the net effect of stimulation specific to the OGP structure and nonspecific inhibition associated with the peptide's high positive net charge.

Catalase protection against hydrogen peroxide-induced injury in rat oral mucosa.

Hydrogen peroxide in contact with the oral mucosa induces vesicle formation and ulceration. This study assesses the protective effect of catalase against the hydrogen peroxide insult in the rat tongue mucosa. In addition, we report a detailed histologic characterization of the mucosal injury. Four repetitive applications of 30% hydrogen peroxide at 15 minute intervals produced a vast edema of the submucosal tissue including the tongue muscles that was seen immediately after the last application. Histomorphometric measurements indicated a substantial regression of the edema 1 day later with the appearance of a very large ulceration lined by a pyogenic membrane. Animals examined 7 days after the hydrogen peroxide administration revealed almost complete healing in terms of absence of edema and renewal of intact epithelial lining. Catalase applied to the tongue before the hydrogen peroxide fully prevented the pathologic tissue reaction. Thus testing of the clinical feasibility of catalase in protecting the oral mucosa against hydrogen peroxide-induced injury is strongly indicated.

Ontogenesis of ultrastructural features during osteogenic differentiation in diffusion chamber cultures of marrow cells.

Three stages of osteogenic differentiation can be identified in in vivo diffusion chamber cultures (DCC) of unselected marrow cells, namely, proliferation, differentiation, and maturation (mineralization). These stages were characterized correlatively by in situ differential cell counts, alkaline phosphatase activity, and mineral accumulation. In the present study, the ultrastructure of marrow cell DCC was examined after incubation for 3-21 days. Features characteristic of osteoblastic and chondroblastic differentiation were first noted in 12 day DCC. Sites of osteoblastic differentiation showed cell-cell contacts associated with an increased cell density. The osteoblastic cells had long processes and were embedded in matrix with prominent fiber bundles reminiscent of collagen type I. The chondroblastic cells appeared solitary in areas of lesser cell density. By contrast to the long osteoblastic cell processes, they had short plasmalemmal projections and the matrix surrounding them contained single, thin, short fibers reminiscent of collagen type II, as well as proteoglycan granules. Both cell types showed prominent cytoskeletal elements, rough endoplasmic reticulum, and Golgi. One finding, previously unnoted in differentiating osteogenic cells, was mitochondria with condensed cristae that represent an increased rate of energy metabolism. These mitochondria were particularly abundant in the differentiation stage and declined as the cultures matured. These findings, together with previous reports in the epiphyseal growth plate, suggest that mineralization is associated with an optimal level of energy metabolism rather than extreme hypo- or hyperoxia. The set of ultrastructural parameters defined here in the marrow cell DCC may serve as useful markers for cells undergoing osteogenic differentiation.

Pattern of gene expression following rat tibial marrow ablation.

Following injury to bone marrow there is a phase of osteogenesis in which bone trabeculae replace the initial blood clot and fill the marrow cavity. The newly formed bone is subsequently fully resorbed by osteoclasts and normal bone marrow is restored. In this study we correlated the morphologic events with the pattern of gene expression that defines this sequence. Following marrow ablation, the trabecular bone volume in the affected section of the marrow cavity increased from control to 27% at day 6, declined to 18% at day 8, and eventually returned to control levels at day 14. Osteoblast number increased up to day 6 and declined substantially by day 8, but the number of osteoclasts peaked between days 8 and 10. Histologic analysis of alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activity correlated with the observed cellular changes. Northern blot analysis of the levels of AP, osteocalcin (OC), and osteopontin (OP) mRNA shows a specific pattern of regulated gene expression, with AP mRNA maximal at day 6, OC mRNA very low until days 6-8, and OP mRNA expressed at very high levels throughout. In addition, procollagen alpha 1(I) and alpha 1(III) mRNAs show a regulated pattern of expression, with procollagen alpha 1(I) maximally expressed between days 4 and 10 and procollagen alpha 1(III) expressed at lower levels between days 4 and 6. The mRNA encoding insulin-like growth factor I (IGF-I) was found to be highly expressed between days 5 and 12; however, transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 3 mRNA were only weakly expressed between days 4 and 10. These data demonstrate a temporal pattern of gene expression consistent with the observed morphologic profile, identify changes in growth factor mRNA that may be related to this repair process, and suggest that this is a suitable model for studying in vivo a synchronized sequence of bone formation and resorption at a well-defined anatomic site.