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.

Regenerating bone marrow produces a potent growth-promoting activity to osteogenic cells.

It is well documented that injury to bone marrow is followed by an osteogenic phase that precedes the complete tissue regeneration. We have recently shown that postablation healing of bone marrow in rat tibiae is associated with a systemic increase in osteogenesis. It was hypothesized that a growth factor(s) with an effect on osteogenic cells is produced in the healing limb, is transferred to the blood circulation, and enhances osteogenesis systemically. To test growth factor production, healing bone marrow-conditioned medium was prepared with tissue separated from rat tibias during the osteogenic phase and assayed for enhancement of mitogenic activity in culture of osteogenic rat osteosarcoma cells (ROS 17/2). Partial purification of healing bone marrow-conditioned medium-derived growth factor(s) consisted of gel filtration on Sephadex G-25, boiling, chromatography on heparin-Sepharose, and gel filtration on Sephadex G-75. Mitogenic activity eluted in the void volume of the Sephadex G-25 column (mol wt greater than 5,000). Potent activity resolved from heparin-Sepharose with PBS, and on filtration by Sephadex G-75 this activity recovered in 3 peaks with mol wt estimates of 35,000, 19,000, and less than 10,000. The partially purified factor also showed considerable stimulatory effect on DNA synthesis in osteoblastic fetal rat calvarial cells and on in vitro elongation of fetal long bone; it had only a small effect on nonosteoblastic ROS and fetal rat calvarial cells. These data indicate that healing bone marrow produces growth factor activity with a preferential effect on osteogenic cells. It is suggested that local growth factors have a role as mediators in the sequence of events whereby bone marrow expresses its osteogenic potential. During postablation healing of bone marrow these factors may also function as systemic promoters to osteogenic cells.

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.

Salivary calculi: ultrastructural morphology and bacterial etiology.

Ultrastructural morphology of 16 salivary calculi was studied by means of transmission and scanning electron microscopy. The external surface was mostly globular or coarse, and on high magnifications and features could be divided into four main groups: a) amorphic calcified deposits covering extensive areas, b) other areas covered with crystals in a variety of arrangements, c) heavy accumulations of calcified rod-like and filamentouslike microorganisms, and d) platelet crystals in juxtaposition to calcified microorganisms in several areas. In most calculi the split area was found to be laminated. It is suggested that microorganisms have an important role in the formation and growth of salivary calculi.

Stimulation of mannose incorporation into rat osteoblastic osteosarcoma cells by parathyroid hormone.

Agents considered to alter cAMP accumulation in bone cells were evaluated for their effects on protein production and sugar incorporation by bone cells. Total protein production or mannose incorporation by cells and in media were measured by the amount of radioactivity incorporated into trichloroacetic-acid precipitable fractions. Incorporation of radioactive leucine into the cellular and secreted proteins was linear over 5 h; radioactive mannose incorporation was linear only up to 2 h. Cells were also incubated with selected agents and radioactive mannose or leucine for 2 h. Addition of parathyroid hormone (PTH), isoproterenol or dibutyryl cAMP to osteoblasts resulted in a significant increase in mannose incorporation; dibutyryl cGMP and butyric acid had no effect on mannose incorporation. None of the agents altered total protein production. Thus PTH stimulates mannose incorporation into osteoblasts and this effect is, at least in part, modulated by cAMP.

Enhancement by extracts of mineralized tissues of protein production by human gingival fibroblasts in vitro.

Non-confluent cell cultures were exposed to both guanidine and guanidine-EDTA extracts of cementum, dentine and alveolar bone, at concentrations from 2 to 50 micrograms/ml for 48 h. The cells were radioactively labelled during the last 24 h. Total protein production was measured via incorporation of radioactive proline; collagen production was estimated by digestion of the radioactive protein mixture with bacterial collagenase. All guanidine-EDTA extracts elicited statistically-significant increases in total protein production when compared to controls. At 50 micrograms/ml of extract, the increase in protein production was 340, 143 and 338 per cent for bone, cementum and dentine, respectively. Similar results were obtained for collagen production. Guanidine-EDTA extracts also stimulated an increase in the production of specific proteins, as ascertained by gel electrophoresis. In contrast, the guanidine extracts had no effect on either protein or collagen production. Thus the functions of gingival fibroblasts can be altered by proteins from associated mineralized tissues. Identification of such proteins and their biological functions would enhance knowledge of the mechanisms that regulate connective-tissue regeneration.

Effect of demineralized bone powder on osteoblast-like cells in culture. A potential rapid quality control assay.

Demineralized bone powder (DBP) has been shown to induce osteogenesis in a variety of bone defects and extra-osseous sites. Previous investigations have been carried out in animal models which are time-consuming and expensive. We studied the effect of DBP on well-established populations of osteoblast and non-osteoblast-like cells in culture to establish an inexpensive, efficient and reliable assay for bone induction. DBP and BP (non-demineralized powder), of particle size 38-53 microns, were prepared from rat long bones. ROS (rat osteosarcoma) 17/2.8 and ROS 24/1 cell lines were subcultured weekly. For both 17/2.8 (well differentiated) and 24/1 (poorly differentiated) cells, proliferation, i.e. cell count, was significantly greater in DBP enriched medium when compared with control or medium with BP. Cell counts for wells with BP were no different from controls. The increased cell count in DBP-enriched medium was significant on days 2-5 (peak effect 2-3 days). Alkaline phosphatase production reached peak levels after day 3 when proliferation was beginning to taper off. In this study a consistent increase in osteoblast proliferation and alkaline phosphatase production under the influence of DBP was demonstrated. The tissue culture assay for proliferation must now be correlated with bone induction in vivo. In future, the method may be useful for investigating the mechanism of bone induction.

Inflammatory cells and bacteria in pericoronal exudates from acute pericoronitis.

The present work is one in a series of studies carried out to verify the relationship between bacteria and gingival tissues in pericoronitis. Exudates from 6 cases of acute pericoronitis were examined by light and electron microscopy, including ultrathin sections and negative staining. While bacterial phagocytosis was prevalent in all the exudates studied, spirochetes, which were the predominant microorganisms, were not observed being phagocytized by PMNs or macrophages. The presence of spirochetes in pericoronitis as compared with acute necrotizing ulcerative gingivitis is discussed.

The diagnostic value of computed tomography in temporomandibular joint synovial chondromatosis.

Synovial chondromatosis of the temporomandibular joint was diagnosed and location of dens particles was assessed by means of computed tomography. Based upon the data collected, the joint was operated conservatively and most of the calcifications were removed leaving the patient symptom free. The importance of an accurate preoperative diagnosis of synovial chondromatosis by use of CT scan is stressed in view of numerous unnecessary condylectomies and parotidectomies reported in the literature.

Iatrogenic exfoliation of teeth by the incorrect use of orthodontic elastic bands.

Four cases of improper use of elastic bands for minor orthodontic treatment are described. For all the patients, the treated teeth were damaged and local surgical intervention was necessary. To prevent complications when elastic bands are being used, the dentist must provide stabilization of the elastic bands by brackets, bands, or bonded attachments, inform the patient about the possibility of losing the rubber elastic band, and mark the elastic rubber by radiopaque material. Every effort must be made to preserve the damaged anterior teeth by fixation and surgical treatment.

[The overlooked chin trauma in children].

UNLABELLED: Children seldom experience major facial trauma. However, minor injuries such as facial lacerations, abrasions and dental trauma are more common and naturally get less attention. Children with TMJ trauma usually present with pain, limited jaw movement and even malocclusion. However, these findings are not always recognized and sometimes get overlooked when chin laceration is the only sign. Plain x-rays can contribute to the diagnosis but in many cases are inconclusive and are difficult to interpret. Often, there is a diagnostic dilemma, especially when the clinical findings are not clear and normal occlusion is hard to restore. We present 3 out of 12 children in whom we diagnosed condylar head fracture following minor facial trauma. All children had normal physiological occlusion. Most of them had mild clinical signs such as limited mouth opening, pain and tenderness upon palpation of the joint. Routine plain films were not conclusive. The diagnosis was made or confirmed with CT scan. Early diagnosis of TMJ fracture in children is mandatory for the prevention of long term functional and esthetically debilitating sequela. CONCLUSION: A thorough clinical examination and normal occlusion do not rule out condylar head fracture after chin trauma. Plain films are of limited value in the diagnosis of such fractures. CT should be considered for the evaluation of these children.

[Maxillofacial gunshot injuries in hostility activities in 2000-2003].

The management of facial gunshot wounded patients requires the coordination of multiple surgical disciplines to optimize the functional and cosmetic outcome while minimizing overall morbidity and mortality. All reports indicate that early management of these patients must focus on the basis of resuscitation, with major attention given to the status of maintaining the airway, hemostasis and hemodynamic resuscitation. Subsequent management of these patients becomes more controversial in terms of surgical reconstruction: immediate or delayed. Involvement of the use of suicide bombers, the type of weapons used in the last 2 years were different than previously described. The injuries were inflicted by explosives mixed with nails bolts and stones, which caused different kinds of wounds ever described. Numerous series in the literature advocate early aggressive intervention of one-stage reconstruction of all involved structures. However, several series published in the literature favor a more conservative approach, some of them even advocating non-operative management of these injuries. We concluded that a delay in final reconstruction of facial fractures in the critically ill patient has an acceptably low complication rate and may be advantageous in decreasing operative risk. A carefully planned reconstruction schedule is required to achieve satisfactory function and appearance. It is our purpose in this report to indicate the difference of the gunshot wounds in the last 2 years of the hostilities in this part of the world and emphasize the function of the Oral & Maxillofacial Surgeons in treating these patients.

The basal cell nevus syndrome associated with cleft lip and cleft palate: report of case.

A case of basal cell nevus syndrome with multiple keratocysts, cleft lip and cleft palate, calcification of the falx cerebri, and late appearance of basal cell carcinoma is described.

Modification of the muco-periosteal flap design in anterior maxillary osteotomies.

A modification of the mucoperiosteal flap planning for anterior maxillary osteotomy operations is suggested. This approach involves a total reflection of the labial mucoperiosteal flap, with the advantages of direct vision of the operative field thus providing easy and precise execution of the operative bony cuts, avoiding damage to the adjacent teeth and reduction of operating time. Sufficient blood supply to the mobilised segment is maintained through the palatal blood vessels. Corrective periodontal surgery may also be carried out at the same time.

Resorption of the crown in impacted maxillary canine. A clinical, radiographic and histologic study.

Thirty-six maxillary impacted canines which showed coronary resorption were selected out of 252 impacted canines. Clinical, radiographic and histologic observations were performed. In the majority of the cases (31) a local inflammatory factor was present and only in five cases was the resorption idiopathic. The incidence of resorption was highest in the later decades of life. In all cases bone replacement was found.

Lymphosarcoma of the mandible associated with macroglobulinemia of Waldenström.

Waldenström macroglobulinemia is a rare progressive immunoproliferative disorder involving lymphocytes and occasionally plasma cells. Unlike the multiple myeloma, this disease has minimal osseous lesions. Lesions in the jaws are very rare. This paper presents a case of lymphosarcoma in the mandible in a 68-year-old woman, who was diagnosed as suffering from Waldenström macroglobulinemia. The possibility of immunosuppression by Leukeran as an aetiologic factor causing the bone lesions is suggested.

Further characterization of osteogenic-cell growth promoting activity derived from healing bone marrow.

During its osteogenic phase, post-ablation regenerating bone marrow produces bone promoting activity to osteogenic cells. In the experiments reported, activity derived from (rat) healing bone marrow conditioned medium (HBMCM) after boiling was analyzed using chromatography on heparin-Sepharose. The activity in HBMCM was shown to be divided among at least six independent activities that stimulated DNA synthesis rates is osteogenic rat osteosarcoma (ROS) cells. Three activities resolved when heparin-Sepharose was washed isocratically with phosphate buffered saline. Two of these were resistant to reduction and acidification and their effect was considerably more potent in osteogenic than non-osteogenic ROS cells. Three additional activity peaks recovered when the heparin-Sepharose column was pumped with an NaCl gradient. Two of them eluted at 0.3 and 0.65 M NaCl, affected osteogenic and non-osteogenic ROS cells to a similar extent and may be attributed to platelet-derived growth factor. A third peak, resolved at 1.2 M NaCl, implies the residual activity of acidic fibroblast growth factor that persisted after boiling of the conditioned medium. It is concluded that the activity profile of HBMCM reflects the in vivo situation where the osteogenic phase of marrow regeneration is probably regulated by multiple growth factor species.

Radiographic and clinical manifestations of the impacted mandibular third molar.

Observations were made on 200 impacted mandibular third molars that were removed from 200 unselected patients under local anesthesia. Sixty percent of the teeth were found to be in apparent relationship with the inferior dental canal: 19 % in true relationship and 41 % in superimposition. The vertical impacted molars were mainly found to be in true relationship with the inferior dental canal. On comparison of the clinical postoperative diagnosis with the radiographic preoperative findings an error of only 0.8 % was shown. Most of the molars showed fully developed roots in the third decade of life. Most of the teeth in this series were vertically impacted having mostly fully developed roots. There was a significant correlation between the patients suffering from pain, trismus, and swelling. Swelling and trismus were the mostly affected postoperative sequelae.