Identifying maternal deaths in developing countries: experience in Jamaica.

Multiple sources were used to identify maternal deaths and their causes in a study carried out in Jamaica. These sources of information included a review of all deaths of women aged 12 to 49 years and included those occurring in hospitals (on maternity, surgical and medical wards and in casualty departments); reported to coroners' offices and the police; on whom post-mortems were carried out at hospitals, public morgues and for the Ministry of National Security; obtained from interviews with public health staff in all parishes and which were registered with the Registrar General's Department. Some 193 maternal deaths were identified giving a maternal mortality rate of 10 per 10,000 live births. No one source independently identified all maternal deaths. Hospital in-patient records yielded 133 deaths (69%), death certificates 74 (38%). Deaths due to certain causes were far more likely to be identified from particular sources eg those due to clinical mismanagement (complications of anaesthesia and blood transfusion) from hospital in-patient records; while deaths from ruptured ectopic pregnancy were more likely to come from coroners', police and morgue records. It is concluded that using multiple sources to identify maternal deaths in developing countries is an effective method to identify all maternal deaths.

CGRP-beta unlike CGRP-alpha has no osteogenic stimulatory effect in vitro.

The purpose of this study is to test whether CGRP-beta has an osteogenic stimulating effect, as does CGRP-alpha, on rat bone marrow cells in vitro. CGRP-beta in different doses was added daily to bone marrow white cells, which were harvested from rats, then seeded onto a previously prepared layer of fibroblasts. CGRP-alpha in different doses was used as a positive control. Fourteen days after the start of the experiment, there was no statistical difference in the number of bone colonies between the control and CGRP-beta dishes. The CGRP-alpha dishes demonstrated an increase in the number of colonies with an increase of peptide dose.

Neurogenic substance P stimulates osteogenesis in vitro.

Previous studies have shown that there is colocalization of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers in bone, periosteum and bone marrow. Because SP may also possibly play a role in bone formation, we decided to test whether it has an osteogenic stimulating effect on developing bone in vitro. To this end, 0.4, 4 and 40 micrograms/ml of SP in BGJb medium was added daily to 3 million light density (LD) bone marrow white cells which were separated by Ficoll-Paque density gradient separation then seeded onto a previously prepared fibroblast feeder layer in Petri dishes. Seven days after adding SP, in the control without SP there were 2 bone colonies; with 0.4 micrograms of SP there were 3 colonies; with 4 micrograms there were 5 colonies; with 40 micrograms there were 7 colonies. In addition, there was an increase in the size of bone colonies in the SP-added group. The results indicated that SP had a dose-related osteogenic stimulating effect. The increase in the number and size of bone colonies by SP was probably caused by stimulating stem cell mitosis, osteoprogenitor cell differentiation or osteoblastic activity.

The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide.

Silverman and Kruger (Somatosens. Res. 5(2):157-175; 1987) reported that sensory nerve fibers of the dental pulp secrete calcitonin gene-related peptide (CGRP). These are localized exactly where secondary or tertiary dentin calcification occurs. Recently we found that CGRP has an osteogenic stimulating effect by increasing the number and size of bone colonies in vitro. The purpose of this study is to test whether there is a relationship between the effects of different doses of CGRP and bone colony numbers and/or size. Rat CGRP in different dosages (0.4, 4 and 40 micrograms/ml in BGJb medium) was added daily to 3 million light density (LD) bone marrow white cells which were harvested from adult Sprague-Dawley rats with the Ficoll-Paque density gradient separation method, then seeded onto a previously prepared feeder layer of fibroblasts in Petri dishes. Seven days after adding CGRP, in the controls without CGRP there were 2 bone colonies; with 0.4 microgram of CGRP there were 4 colonies; with 4 micrograms of CGRP there were 6 colonies; with 40 micrograms there were 9 colonies, indicating there was a significant increase in number of bone colonies with an increase in dose of CGRP between individual groups, respectively (p less than 0.0005 and p less than 0.0001). In another experiment, intravenous injection of 10 micrograms of rCGRP/kg body weight was performed two hours before surgery. LD bone marrow white cells were collected and seeded onto a feeder layer in Petri dishes exactly as described above.(ABSTRACT TRUNCATED AT 250 WORDS)

A technique for the removal of junctional epithelium from the epithelial attachment.

A technique utilizing sodium deoxycholate to remove junctional epithelial cells from the epithelial attachment zone is described. Light and electron micrographs show a similarity in size and appearance between the extracellular attachment substance in controls and the extracellular substance which remains on cementum previously in contact with junctional epithelium.

Osteogenesis enhanced by chitosan (poly-N-acetyl glucosaminoglycan) in vitro.

Chitosan, with a chemical structure similar to hyaluronic acid, has been implicated as a wound healing agent. The purpose of this research was to evaluate the effect of chitosan on osteoblast differentiation and bone formation in vitro. Mesenchymal stem cells were harvested from fetal Swiss Webster mice calvariae prior to osteoblast differentiation and calcification (12 to 13 days in utero). Stem cells were seeded into 6-well culture plates at a density of 350,000 cells per well. Using this model, it was possible to quantify the influence of chitosan on osteoprogenitor differentiation and osteogenesis. Experimental wells were pretreated with 200 microliters chitosan (2 mg/ml in 0.2% acetic acid vehicle). Control wells were pretreated with 200 microliters vehicle (0.2% acetic acid) or remained untreated. Cells were allowed to grow under optimal conditions for 14 days. Cell cultures were fixed with glutaraldehyde and stained with Von Kossa stain to identify bone forming colonies. Positive staining colonies were identified and counted under light microscopy. Histologic cross-sections of representative positively stained colonies identified osteoblasts and confirmed bone formation. Examination of control wells revealed 3.6 +/- 0.6 colonies per well while experimental wells revealed a significantly greater average of 6.2 +/- 1.2 colonies per well (P < or = 0.01). Computer-assisted image analysis of the average area of bone formed by control colonies was 0.34 +/- 0.09 (relative units) while that of experimental colonies was 0.39 +/- 0.06 (relative units) per average bone forming colony. The difference in mean size (control versus chitosan bone forming colony) was not statistically significant (P = 0.4691). The results of this in vitro experiment suggest that chitosan potentiates the differentiation of osteoprogenitor cells and may facilitate the formation of bone.

Bacteriostatic effects of hyaluronic acid.

BACKGROUND: This investigation is one of a series of projects seeking to ascertain whether hyaluronic acid (HA) is therapeutically effective in tissue regeneration procedures. The rationale for these investigations is to test the hypothesis that HA can serve as a bioabsorbable carrier for other substrates as well as itself actively promote the regeneration of tissue. METHODS: In this paper, we report on the bacteriostatic and bactericidal properties of 3 molecular weight formulations of recombinant HA (low, 141 kD; medium, 757 kD; and high, 1,300 kD) on selected oral and non-oral microorganisms in the planktonic phase. Three concentrations of each HA formulation were screened, 0.5, 1.0, and 2.0 mg/ml, using a standard broth culture assay. RESULTS: Recombinant HA exerted varied bacteriostatic effects on all the bacterial strains tested depending on its molecular weight (MW) and concentration. The high concentrations of the medium MW HA had the greatest bacteriostatic effect, particularly on the Actinobacillus actinomycetemcomitans, Prevotella oris, Staphylococcus aureus, and Propionibacterium acnes strains. The 1.0 mg/ml concentration of high MW HA had the greatest overall bacteriostatic effect, inhibiting the growth of all 6 bacterial strains tested. Among the bacterial strains studied, HA was found to have no bactericidal effects, regardless of concentration or molecular weight. CONCLUSIONS: The results of this study suggest that HA in the MW range of 1,300 kD may prove beneficial in minimizing bacterial contamination of surgical wounds when used in guided tissue regeneration surgery.

In vitro comparison of aged and young osteogenic and hemopoietic bone marrow stem cells and their derivative colonies.

The purpose of this in vitro study was to determine whether there were differences in the number and size of osteogenic and hemopoietic colonies derived from bone marrow stem cells of aged and young adult male Sprague-Dawley rats. Using a Ficoll-Paque gradient, stem cells were harvested from aged male rats 18 to 22 months old and young adult males 55 days of age. Single cell suspensions from the red marrow of the long bones were cultured 14 days in vitro and subsequent colonies were assessed by light microscopy for number and size. A computerized histomorphometric linear measuring system was utilized to assess colony area in square millimeters. The results clearly show that young animals have a statistically significant increased cellular potential for osteogenic and hemopoietic colony formation. Cultures from aged animals showed an average formation of 0.45 +/- 0.6863 osteogenic colonies while those from younger animals had an average of 3.6 +/- 2.3523 osteogenic colonies per 3 million cells plated. Hemopoietic colonies from aged animal cell cultures numbered 5.25 +/- 2.2449 while those from the young animals averaged 8.23 +/- 3.3601 per 3 million cells plated. The difference in size of the osteogenic and hemopoietic colonies between age groups was not statistically significant. The area of osteogenic colonies derived from aged animals measured 0.1244 +/- 0.0891 mm2, while those derived from the young animals averaged 0.1276 +/- 0.0518 mm2. Hemopoietic colonies from the aged cells measured 0.0759 +/- 0.0514 mm2, while hemopoietic colonies from the young animal cells measured 0.06010 +/- 0.0180 mm2. The results of this study may have implications for consideration in the cellular healing aspects of aged versus young individuals.

Hyaluronan supports recombinant human bone morphogenetic protein-2 induced bone reconstruction of advanced alveolar ridge defects in dogs. A pilot study.

BACKGROUND: Prosthetic-driven implant dentistry requires predictable procedures for alveolar ridge augmentation. The objective of this pilot study was to evaluate bone regeneration in mandibular, full-thickness, alveolar ridge, saddle-type defects following surgical implantation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a novel hyaluronan (HY) sponge carrier. This sponge was fabricated from auto-crosslinked HY. METHODS: Alveolar ridge defects (approximately 15 x 10 x 10 mm), 2 per jaw quadrant, were surgically prepared in each of 3 young adult American fox hounds. Four defects were immediately implanted with rhBMP-2/HY. Three defects were implanted with rhBMP-2 in an absorbable collagen sponge (ACS) carrier (positive control). The rhBMP-2 solution (1.5 ml at 0.2 mg/ml) was soak-loaded onto the HY and ACS sponges. Three defects were implanted with HY sponges soak-loaded with buffer without rhBMP-2 (negative control), while 2 defects served as surgical controls. The animals were euthanized at 12 weeks postsurgery for histometric analysis. RESULTS: Clinically, alveolar ridge defects receiving rhBMP-2/ACS exhibited a slight supracrestal expansion, while defects receiving rhBMP-2/HY were filled to contour. In contrast, the HY and surgical controls exhibited ridge collapse. rhBMP-2/HY-treated defects exhibited a dense bone quality without radiolucent regions observed in defects treated with rhBMP-2/ACS. The histometric analysis showed 100% bone fill for the rhBMP-2/ACS defects and 94%, 58%, and 65% bone fill for the rhBMP-2/HY, HY, and surgical control defects, respectively. CONCLUSIONS: The conclusions are based on data from 2 of 3 animals in the study. In one animal, no response to rhBMP-2 was observed with either carrier, and the animal may have been a non-responder of unknown nature. With this limitation, the observations herein suggest that: 1) HY supports significant bone induction by rhBMP-2; 2) the rhBMP-2-induced bone assumes qualities of the immediate resident bone; 3) HY alone exhibits no apparent osteoconductive potential; and 4) HY appears to resorb within a 12-week healing interval in the absence or presence of rhBMP-2. Thus, HY appears to be a suitable candidate carrier for rhBMP-2.

Healing and repair of osseous defects.

This article has discussed most of the modalities currently in use for the filling of bony defects. No one modality or combination of methods or materials has yet surfaced as the definitive means of treatment for filling these defects.

Ultrastructural in vitro characterization of a porous hydroxyapatite/bone cell interface.

In order for the hydroxyapatite implant material interface to new bone to be characterized, osteogenic mouse calvarial mesenchymal cells were grown in vitro in contact with a porous hydroxyapatite (PHA). After the mesenchymal cell culture was incubated for 12 to 13 days, the resulting tissue-containing bone colonies were fixed, embedded, sectioned, and stained for microscopic evaluation. Light and transmission electron microscopy (with conventional staining) and phosphotungstic acid cytochemistry were used to explore and record the optical microscopic and ultrastructural interfaces at the hydroxyapatite surface. Osteoblasts, fibroblasts, bone, and cartilage were observed and photographed at the implant surface. Osteoblasts found in conjunction with well-developed collagen, matrix vesicles in the extracellular matrix, and newly formed hydroxyapatite crystals on the PHA surface confirmed the beginning of woven bone formation. Collagen fibers were observed directly in contact with the PHA when osteoblasts were present. Polysaccharides were localized among the collagen fibers in the implant-cell extracellular space, indicating a rich complex carbohydrate layer in relation to the collagen of immature bone. Fibroblasts and chondroblasts at the implant surface secreted no collagen, but an amorphous layer was visible between the fibroblasts and the implant surface. When polysaccharides were stained, an electron-dense film appeared where the amorphous layer came into contact with the implant material. Collagen was secreted from the cell surface furthest from the implant. Osteoblasts and fibroblasts/chondroblasts, when surrounding PHA, seem to take on two different interfacial functions: Osteoblasts secrete collagen in a bone-initiating extracellular matrix with carbohydrates at the implant surface, whereas fibroblasts/chondroblasts appear to attach to the implant with a carbohydrate-rich attachment substance, but with no collagen at the interface. This study confirms in vivo data that PHA is a viable implant material because it is biocompatible and, unlike several other materials, appears to stimulate, or at least to permit, osteogenesis.

Effect of hyaluronan on calcification-nodule formation from human periodontal ligament cell culture.

The periodontal ligament (PDL) has been found to house progenitor elements which may give rise to a new periodontium when properly triggered. Complex molecular mechanisms are involved in guiding such cells towards their final regenerative task. Hyaluronan (HA), an extracellular matrix molecule abundantly present during early tissue development, is usually found at high concentrations in all adult mammalian PDL tissue. In this study, PDL cells were cultured on HA coated experimental plastic Petri's dishes (E.D.) and mineralized-nodules formation, at the microscopical level, was evaluated in comparison to HA-free control dishes (C.D.) after 21 days. Little colony formation occurred in presence of HA, whereas CD showed normal cell confluence with mineralization of nodules even at 10 days. However, when harvesting was performed from both dishes after only 12 hours, before cell aggregation occurred, cell confluence and nodule formation could be seen with no apparent difference between test and control. This study shows that, with respect to PDL cells, hyaluronan seems to inhibit cell adhesion, although with no interference with cell aggregates calcifying potentials. This latter finding may: a) provide further insight in analyzing the properties of extracellular matrix substrates in the PDL environment, b)guide research to possible explanations on how the PDL maintains its characteristics of uncalcified tissue and c) lead to theorize regarding utilization in tissue regeneration. (Journal of Applied Biomaterial & Biomechanics 2003; 1: 84-90).

Ultrastructural localization of alkaline phosphatase in initial intramembranous osteogenesis.

Developing fetal calvariae in which calcification of woven bone has been initiated, have been decalcified with formic acid, buffered with citrate at pH5 to remove all traces of calcium and phosphate from the tissue. The decalcified bone was then incubated in medium containing sodium-beta-glycerophosphate, an artificial substrate for alkaline phosphatase. The Hugon and Borgers-modified Gomori reaction revealed the sites of A-Pase activity as electron dense deposits of lead phosphate. 23,24 One site of A-pase localization was in Golgi vesicles. Another site, at the cell membrane, indicated that A-Pase was apparently secreted from the osteoblasts into the calcifying matrix. A strong reaction in the matrix vesicles and at the periphery of bone nodules (vesicles) indicated that A-Pase was localized ultrastructurally in the precise zones of calcification during initial intramembraneous ossification.

Osteogenesis and leukopoiesis within diffusion-chamber implants of isolated bone marrow subpopulations.

The developmental potential of isolated rabbit bone marrow cell populations was examined following autogeneous implantation into diffusion chambers. After 4 weeks, the implants were harvested and processed for light and electron microscopy. More total tissue was formed in abdominally implanted chambers than in corresponding femoral chambers. Two of the separated marrow cell populations produced significantly greater amounts of fibrous connective tissue, cartilage, and bone than did whole-marrow controls. These two populations, which were defined by density gradients of 1.050-1.055 gm/cm3 and 1.064-1.067 gm/cm3, consistently produced a fibrous connective tissue nodule in which were found dispersed foci of hyaline cartilage and woven bone. The denser population was distinguished further by the presence of leukopoietic foci in several of the implant chambers. Cartilage foci were found predominantly towards the center of the tissue nodules, whereas bone was dominant towards the periphery. Blood vessels, osteoclasts, bone remodeling, and mature lamellar bone were found only in those chambers which had been penetrated by the host's vascular system. The results indicate that 1) normal marrow tissue contains two separate osteoprogenitor cell populations; 2) these two progenitor populations represent separate osteogenic and chondrogenic capabilities; 3) one of these populations possesses a leukopoietic as well as an osteogenic potential; and 4) a competent vascular system is essential for the remodelling of bone into mature bone organs.

Peripheral blood mononuclear cells develop into multinucleated osteoclasts in tissue culture.

BACKGROUND: Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood. The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding mononuclear cells from peripheral blood into established explants of fetal mouse calvaria in vitro. METHODS: In order to utilize osteoclast-free bone, the fetal calvariae were obtained from 13-14-day pregnant Swiss Webster mice and cultured in BGJb medium for 9 days. At day 9, peripheral blood mononuclear cells were isolated as a light density layer from adult Swiss Webster mice with the Ficoll-Paque density gradient separation method and co-cultured with the osteoclast-free, fetal mouse calvaria. RESULTS: After 10 days of co-culture, multinucleated cells, which have all the characteristics of osteoclasts, were found in juxtaposition to seams of woven bone. Two multinucleated osteoclasts per one million light density peripheral blood mononuclear cells were found in the experimental group; none were found in the mononuclear cell-free control group. CONCLUSIONS: Peripheral blood mononuclear cells can give rise to multinucleated osteoclasts in developing bone in vitro but will not develop without bone.

Calcitonin gene related peptide enhances bone colony development in vitro.

Recent evidence suggests that sensory nerve fibers of the dental pulp secrete calcitonin gene related peptide alpha exactly where secondary or tertiary dentin is mineralized. In addition, calcitonin gene related peptide raises the level of cyclic adenosine monophosphate in osteoblasts, indicating a potential effect on secretory activity in bone. Because calcitonin and calcitonin gene related peptide are formed from the same gene, the authors tested whether calcitonin gene related peptide or calcitonin has an osteogenic potential in vitro. To this end, 0.01, 0.1, and 1 microg/ml of salmon calcitonin or rat calcitonin gene related peptide in Bigger, Gwatkin, Jackson b medium was added daily to 3 x 10(6) rat light density bone marrow leukocytes that were separated with the Ficoll-Paque density gradient separation method, then seeded on a previously prepared fibroblast layer in Petri dishes. After 7 days, the number and size of bone colonies formed in the calcitonin gene related peptide (0.1 or 1 microg/ml) added group was significantly greater than that of the control group. There was no statistically significant difference between the calcitonin added and control groups. Calcitonin gene related peptide has an osteogenic stimulating effect, either by stimulating stem cell mitosis or osteoprogenitor cell differentiation (or both), whereas salmon calcitonin has no effect.

Osteoclast formation in vitro from bone marrow mononuclear cells in osteoclast-free bone.

Recent evidence points to the fact that osteoclasts are derived from mononuclear cells of hematopoietic bone marrow. In this study we have examined the formation of osteoclasts from mononuclear cells in vitro. The mononuclear cells were isolated after 7 days from cultures of mouse bone marrow cells. The isolated cells were co-cultured with osteoclast-free, fetal-mouse calvaria. After 10 to 14 days of co-culture, multinucleated cells which have all the characteristics of osteoclasts were found in juxtaposition to seams of woven bone. These data strongly suggest that bone marrow mononuclear cells, when suitably induced, can give rise to osteoclasts in vitro.