Effect of bone morphogenetic protein-2, demineralized bone matrix and systemic parathyroid hormone (1-34) on local bone formation in a rat calvaria critical-size defect model.

AIM: To determine the potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) soak-loaded on to an absorbable collagen sponge (ACS) to induce local bone formation compared with the clinical reference demineralized bone matrix (DBM) and to investigate potential additive/synergistic effects of exogenous parathyroid hormone (PTH). METHODS: Critical-size (8 mm), through-through calvaria osteotomy defects in 160 adult male Sprague-Dawley rats were randomized to receive one of eight interventions: rhBMP-2/ACS, DBM, ACS, or serve as controls (empty defects) combined or not with systemic PTH. Ten animals from each group were followed for 4 and 8 wks for radiographic and histometric analysis. Multivariable analysis was used to assess the effect of experimental intervention and healing time on local bone formation. RESULTS: In the multivariable analysis, rhBMP-2/ACS exhibited significantly greater histologic bone formation than control (ß ± SE: 54.76 ± 5.85, p < 0.001) and ACS (ß ± SE: 9.14 ± 3.31, p = 0.007) whereas DBM showed significantly less bone formation than control (ß ± SE: -32.32 ± 8.23, p < 0.001). Overall, PTH did not show a significant effect on bone formation (ß ± SE: 2.72 ± 6.91, p = 0.70). No significant differences in histological defect closure were observed between 4 and 8 wks for all but the control group without PTH. CONCLUSION: rhBMP-2/ACS significantly stimulates local bone formation whereas bone formation appears significantly limited by DBM. Systemic application of PTH provided no discernible additive/synergistic effects on local bone formation.

Bisphosphonate-ciprofloxacin bound to Skelite is a prototype for enhancing experimental local antibiotic delivery to injured bone.

BACKGROUND: The risk of osteomyelitis after open bone fracture may be reduced by locally applied antibiotics. ENC-41-HP (E41), which comprises ciprofloxacin linked to a 'bone seeking' bisphosphonate, loaded on to carrier Skelite calcium phosphate granules (E41-Skelite) has favourable in vitro characteristics for application to wounded bone. This study assessed E41-Skelite in a rat model of acute tibial osteomyelitis. METHODS: Mechanically induced tibial troughs were contaminated with approximately log10 4 colony forming units (c.f.u.) of Staphylococcus aureus (Cowan 1 strain) 'resistant' to E41 (minimum inhibitory concentration 8-16 microg/ml), lavaged and packed with Skelite alone, or with E41-Skelite slurry. Animals were killed at 24 h (n = 62), 72 h (n = 46) or 14 days (n = 12), and each tibia was assessed for S. aureus load (c.f.u./g tibia) and histological appearance (14 days only). RESULTS: At 24 and 72 h, the tibias of rats treated with E41-Skelite (n = 54) had a significantly lower mean (s.e.m.) load of S. aureus than animals that received Skelite alone (n = 54): log10 3.6(0.2) versus 6.4(0.1) c.f.u./g respectively at 24 h (P < 0.001, Mann-Whitney rank sum test) and log10 4.4(0.2) versus 6.6(0.1) c.f.u./g at 72 h (P < 0.001). At 14 days, E41-Skelite-treated tibias had fewer bacteria, no signs of osteomyelitis and histological signs of healing. CONCLUSION: E41-Skelite, a prototype granulated topical antibiotic delivery system, reduced the development of infection in experimental bone wounds.

Effect of the wicking behavior of multifilament sutures.

The purpose of this study was to compare the wicking propensity of multifilament sutures. Dexon II, Vicryl, and black silk suture (BSS) were dipped in saline or soaked for 48 h, then suspended on a microscope slide. Fluorescein isothiocyanate-dextran (FITC-D) was placed at the suture mid points, and its movement was observed using fluorescence microscopy. The experiment was repeated, replacing the FITC-D with mixture of S. salivarius and saline, incubating the suture specimens in culture medium, and evaluating microbial growth. Dipped sutures showed FITC-D movement in the Dexon II group only. All 48-h soaked sutures demonstrated FITC-D movement with significant (p < 0.005) differences in mean times: BSS 179 +/- 42 s; Vicryl 120 +/- 26 s; and Dexon II 32 +/- 2 s. Dexon II suture demonstrated wicking of S. salivarius, whereas Vicryl and BSS did not (p < 0.05). These results suggest that BSS and Vicryl sutures do not wick as readily as Dexon II does.

The effect of various intracanal oxidizing agents on the push-out strength of various perforation repair materials.

OBJECTIVE: The purpose of this study was to determine the effect of intracanal oxidizing agents on the strength of materials used to repair root perforations. STUDY DESIGN: Standardized perforations in bovine root samples were repaired with mineral trioxide aggregate (MTA), Super-EBA cement (S-EBA), or intermediate restorative material (IRM). After 7 days, 10 samples from each group were tested for push-out strength with an Instron machine (controls). The remaining samples were immersed in NaOCl, sodium perborate mixed with saline (SPB+S), Superoxol (SO), sodium perborate mixed with Superoxol (SPB+SO), or saline for 7 days to investigate the effect of irrigating and walking bleach compounds on the perforation repair materials. Push-out strength values were compared with those of the dry materials to determine whether any loss of integrity had occurred. RESULTS: MTA was statistically significantly less resistant across conditions to displacement than S-EBA or IRM. IRM was consistent across treatment conditions, whereas S-EBA lost strength when exposed to NaOCl, SPB+S, or SPB+SO. Exposure to SPB+S had the greatest effect on all 3 materials. CONCLUSIONS: IRM performed consistently as a perforation repair material despite exposure to oxidizing agents, whereas MTA was less resistant to dislodgement than either IRM or S-EBA and was more affected than IRM by sodium perborate-containing bleaching solutions.

Adaptation of a dental RadioVisioGraph unit as a laboratory animal research tool.

We have adapted the RadioVisioGraph (RVG), a digital radiography system designed for dentistry, to become a versatile research tool in a small research facility. We have used this modified digital imaging system in our institution to assess bone fractures and ossification in rabbit tibias in which titanium posts were placed in close proximity to one another, to evaluate bone fill in rats with experimental cranial critical-size defects, and to ensure the proper placement of oral gavage tubes in rodents. This method provides instantaneous digital radiographs, thus not requiring a dedicated X-ray suite or film-processing equipment, and reduces scatter radiation by < or =95%. The use of this technology in a small research facility has greatly improved the quality of both the care our animals receive and the research data we obtain.

Antimicrobial activity of several calcium hydroxide preparations in root canal dentin.

The purpose of this study was to evaluate the antimicrobial activity of several calcium hydroxide (Ca(OH)2) preparations in root canal dentin infected with Enterococcus faecalis. Roots of extracted bovine incisors were prepared to standardized cylindrical test specimens of 5 mm in height; the smear layer was removed, and the specimens were incubated for 24 h at 37 degrees C in bacteriological culture medium that contained 7.0 x 10(4) colony forming units per milliliter of E. faecalis. The specimens were mounted in individual 4-mm diameter culture wells, and the test material was applied to fill the canal lumen. There were five treatment groups: group 1, a thick mixture of Ca(OH)2 USP (1.0 g/ml H2O); group 2, a thin mixture of Ca(OH)2 USP (0.1 g/ml H2O); group 3, Pulpdent TempCanal paste; group 4, sterile H2O (positive control); and group 5, 25 dentin specimens in sterile, uninoculated brain-heart infusion broth that were included as negative controls. Quantitative microbiological analysis of dentin at various depths was completed after 24 h. All groups showed a significant (p < 0.001) decrease in numbers of E. faecalis in all depths of dentin compared with the control. Groups 2 and 3 demonstrated significantly greater antimicrobial activity (73%-86% reduction) at all depths of dentin tested compared with group 1 (13%-26%) (p < 0.05). These results suggest that Ca(OH)2 can decrease the numbers of E. faecalis at all depths of dentinal tubules within 24 h and that thin preparations of Ca(OH)2 may be more effective in the elimination of E. faecalis from dentinal tubules than thick preparations.

Effects of nicotine on the strength of attachment of gingival fibroblasts to glass and non-diseased human root surfaces.

BACKGROUND: The aim of this study was to evaluate the effect of nicotine on the strength of attachment of human gingival fibroblast cells to glass and non-diseased human root surfaces. METHODS: Human gingival fibroblast cells (HGF) were trypsinized, suspended in RPMI 1640 medium, and incubated with autoclaved human root and glass sections and nicotine (NIC) concentrations of 0 (control), 25, 50, and 100 ng/ml for 1 week. HGF attached and grew on glass and root surfaces for 4 weeks at all NIC concentrations. HGF cultures were subjected to a rotary shaker machine for 30 minutes to test the strength of attachment of these cells at 100, 150, and 200 rpm. The root and glass sections were examined at 48 hours by light microscopy. RESULTS: Control groups exhibited a monolayer of long, spindle-shaped fibroblasts with a parallel alignment and minimal overlapping. With a concentration of NIC of 50 or 100 ng/ml as well as with increasing "speeds," the number of cells attached to these surfaces decreased dramatically. When 200 rpm was used for both groups at all NIC concentrations, very few HGF remained attached to these surfaces. CONCLUSIONS: This study showed that the nature of cell attachment to either glass or root surfaces is altered by nicotine, and marked detachment was noted when nicotine exposure was coupled with vigorous agitation at different rpm. Marked detachment noted in all specimens at 200 rpm indicates that this speed is excessive for use in subsequent experimentation.

Pluronic polyol effects on human gingival fibroblast attachment and growth.

BACKGROUND: Enhanced speed of human gingival fibroblast (HGF) spreading and attachment, as affected by ionic bonding interactions, may facilitate cell orientation and subsequent collagen synthesis to promote early wound healing. The purpose of this study was to determine the in vitro effects of pluronic polyols, a family of widely used surfactants currently used as drug carriers for antibiotic, anti-inflammatory, and anti-neoplastic agents, on the attachment and growth of human gingival fibroblasts (HGF) to dentin and plastic surfaces using established tissue culture techniques. METHODS: Plastic culture wells containing Eagle's minimal essential media (EMEM) with 10% fetal calf serum and Pluronic F-68 or F-127 in concentrations from 1.2 x 10(-2) to 1.2 x 10(-10) M were incubated with HGF and run in replicates of ten. Attached cells were quantified by measuring the optical density of methylene blue-stained cells. Additional experiments were conducted using human dentin sections as a substrate and Pluronic F-68 or F-127 at a concentration of 1.2 x 10(-8) M. In these experiments, HGF were stained with acridine orange and quantified per unit area of dentin by fluorescence microscopy. RESULTS: Attachment and growth of HGF to both plastic and dentin were significantly increased over serum controls by very low concentrations of Pluronic F-68 and F-127 by 30 minutes, with attachment reaching a plateau at 2 hours. CONCLUSIONS: Pluronic polyols, a family of widely used surfactants, in very low dosages may be beneficial in early postsurgical wound healing by facilitating early attachment and enhancing the growth rate of human gingival fibroblasts.

Determination of periodontal ligament cell viability in long shelf-life milk.

The purpose of this study was to determine the ability of long shelf-life milk to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability on avulsed teeth. PDL cells were plated onto 24-well culture plates and allowed to attach for 24 h. Minimal Essential Medium was replaced with regular pasteurized milk (refrigerated milk), long shelf-life milk (Parmalat), or Save-A-Tooth. Tap water served as the negative control, and Minimal Essential Medium served as the positive control. The tissue culture plates were incubated at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined using a cell proliferation assay (CellTiter 96 AQ Assay) and absorbance read at 490 nm. ANOVA indicated that all media performed significantly better than tap water at all time periods. At 8 h, PDL cell viability in regular pasteurized milk and long shelf-life milk were significantly greater than in Save-A-Tooth (p < or = 0.001). There was no significant difference between regular pasteurized milk and long shelf-life milk at any time period. These results suggest that long shelf-life milk, which has the advantage of not requiring refrigeration, is as effective a storage medium for avulsed teeth as regular pasteurized milk and more effective than Save-A-Tooth.

The microbial proteome database--an automated laboratory catalogue for monitoring protein expression in bacteria.

Laboratories devoted to high-throughput characterisation of purified proteins arrayed via two-dimensional (2-D) gel electrophoresis face an arduous task in maintaining a centralised and constantly evolving record of information relating to the characterisation of proteins and their responses following biological challenges. The Microbial Proteome Database (MPD) has been conceived as an in-house resource for complementing the plethora of genomic databases available for such organisms. The database utilises commercially available software to provide an electronic 'lab book' of information obtained daily from 2-D electrophoresis gels, image analysis packages, protein characterisation methodologies, and biological experimentation. The MPD begins from a single 2-D gel image (a 2-D 'reference map') with clickable spots that link to a 'protein catalogue' (ProtCat) with spot information including protein identity, changes in expression determined under experimental conditions, cellular location, mass, and pI. The entry for each protein then contains further links to gel images corresponding to the presence of the particular protein within different subproteomes (as defined by the pH of narrow- and wide-range immobilised pH gradients or from differential extraction methods used to determine the location of the protein within a functional cell). The database currently contains information from strains of three microbial species (Escherichia coil, Pseudomonas aeruginosa and Staphylococcus aureus) and 32 master gel images. The rapid accessibility of information obtained from microbial proteomes is an essential step towards the integrated analysis of these organisms at the gene, transcript, protein and functional levels and will aid in reducing turnaround times between sample preparation and the discovery of molecules of biological significance.

The effects of intracanal medicaments, fillers, and sealers on the attachment of human gingival fibroblasts to an exposed dentin surface free of a smear layer.

To date there has been very little research into the possible effects of endodontic therapy on regeneration of the lost periodontal attachment. The objective of this study was to examine the effects of endodontic medicaments on fibroblast attachment to dentin surfaces free of a smear layer. Pulp chambers of extracted third molars were filled with one of the following medicaments: gutta-percha with Roth's zinc oxide and eugenol-based sealer, warm gutta-percha with sealer, warm gutta-percha without sealer, calcium hydroxide, formocresol, cotton pellet, or left empty. A predetermined dentin surface area was then inoculated with human gingival fibroblasts at a concentration of 2 x 10(4) cells per ml. The cells were allowed to adhere to the dentin surface for either 4 or 24 hours, then cell attachment was quantified using a methyl-tetrazolium assay. The data were analyzed using a Kruskal-Wallis one-way analysis of variance and Dunn's multiple comparison test. It was determined that fibroblast attachment was significantly reduced when exposed to formocresol or warm gutta-percha without sealer at both the 4 and 24 hour interval (P < or = 0.05). This suggests that the use of formocresol or warm gutta-percha without sealer in a root canal may impede periodontal wound healing and regeneration.

The effect of chlorhexidine irrigation on tensile wound strength.

Chlorhexidine in an alcohol vehicle with flavoring agents has been used as a mouthrinse to reduce plaque accumulation in periodontal surgery patients. The purpose of this study was to evaluate the effects of a chlorhexidine-containing mouthrinse on the early tensile wound strength of healing surgical wounds in the rat. Standardized transdermal incisions were made on each lateral abdominal wall of 40 Sprague-Dawley rats. Wounds were irrigated with 10 ml of 0.12% chlorhexidine or 10 ml of normal saline prior to closure. Animals were sacrificed at 48 hours and 96 hours, and the wound area was excised by a standardized protocol. Wound strength was measured using constant speed tensiometry to determine the tensile strength of the healing incision. Results revealed a significantly reduced tensile wound strength at 48 hours for the chlorhexidine-treated group (127 +/- 18.5 gm) compared to the saline irrigation group (150 +/- 32.3 gm) (P < 0.001). However, by 96 hours a significantly increased tensile wound strength was demonstrated by the chlorhexidine treated group (202.1 +/- 21.7 gm) compared to the saline irrigation group (183.2 +/- 37.3 gm) (P < 0.05). These data suggest that chlorhexidine-containing mouthrinse irrigation of wounds produced a reduced early tensile wound strength, but ultimately resulted in shorter healing time.

Intravenous Pluronic F-127 in early burn wound treatment in rats.

A dramatic improvement in full skin thickness burn wounds in rats treated intravenously with the non-ionic surfactant Pluronic F-127 (F-127) has been demonstrated. In this study the F-127 was given 30 min postburn to simulate conditions encountered in a clinical setting. Anaesthetized male rats (300-320 g) received full skin thickness burns by immersion of the anterior chest wall (8 per cent body surface area in a 70 degrees C water-bath for 12 s). Burn wound area was measured immediately and after 48 h. Thirty minutes after the burn, half the animals received equal volumes (8 ml/kg body wt) of either saline or F-127 (12 mM/l concentration) via the tail vein. The animals autopsied at 48 h showed a significant (P < 0.05) reduction in the degree of wound contraction and the wound appeared grossly less damaged in the F-127-treated animals. Histologically, skin biopsies showed less of the microscopic damage usually associated with full skin thickness burns in the F-127-treated animals than in the saline controls. We also used thermography to measure skin temperature of the burn area at 90 min and 48 h postinjury demonstrating alterations in the F-127-treated animals (P < 0.05). In animals followed for 30 days postinjury, there was a significant (P < 0.01) improvement in the wound closure rates in the F-127-treated animals. These observations show a positive therapeutic effect of F-127 on the inflammatory process in the area of a burn that may improve wound healing.

In vitro attachment of human gingival fibroblasts to endosseous implant materials.

In recent years, the focus of dental implant research has been the nature of the bone-implant interface associated with osseointegration, yet the transgingival portion of endosseous dental implants has received little attention. The purpose of this study was to determine the attachment of human gingival fibroblasts to three different implant materials: commercially pure titanium, non-porous hydroxyapatite, and porous hydroxyapatite. Cell attachment was quantified by radiolabeling gingival fibroblasts with tritiated thymidine and counting attached cells by liquid scintillation following incubation for periods of 20, 40, and 60 minutes. Additional studies coating implant surfaces with fibronectin were also performed. The nature of the implant material itself appeared to affect the number of attached cells. Determined on a surface area basis, fibroblast attachment was greatest to titanium followed by non-porous hydroxyapatite. Porous hydroxyapatite demonstrated the least amount of fibroblast attachment. When incubated with fibronectin at a concentration of 50 micrograms/ml, no increase in the number of cells attached to the various implant materials was observed. A small but statistically significant increase in the number of fibroblasts attached to porous hydroxyapatite at 40 minutes was observed when implant materials were pre-treated with fibronectin.

The effect of chlorhexidine treatment of root surfaces on the attachment of human gingival fibroblasts in vitro.

Chlorhexidine mouthrinse is a widely used adjunct in periodontal therapy due to its bactericidal effects. The effect of this agent on chronic gingivitis and wound healing following surgical therapy in animals and humans has been favorable. The re-establishment of lost connective tissue attachment to the root surface following periodontal therapy is a desirable goal in which the ability of periodontal ligament fibroblasts to reattach to root surfaces of periodontally involved teeth is a critical event. Understanding the effect of chlorhexidine on fibroblast attachment will provide the rationale for its use during the healing phase of periodontal surgery. For this study, impacted third molars were sectioned into 4 pieces. Groups of 10 root pieces were exposed to 0.12% chlorhexidine or saline for 3 minutes followed by a distilled water rinse. The root pieces were incubated with human gingival fibroblasts (HGF) using standard tissue culture techniques for 1, 2, 4, 6, and 8 hours. HGF were prelabeled with 3H-thymidine to a standard specific activity. The surface area of each root piece was determined and the attached cells quantified by using scintillation spectroscopy. The number of cells per unit area was then calculated and the data expressed as cells/mm2. The repeated measures design was statistically analyzed by repeated measures analysis of variance. There was a significant difference between the number of attached cells in the chlorhexidine and the control groups (P less than 0.001). Exposure of root surfaces to chlorhexidine significantly inhibits subsequent fibroblast attachment which may interfere with regeneration of the periodontium. Hence, the data suggest that efforts should be made to minimize chlorhexidine contact with the root surface with physical barriers.

Preliminary results suggesting exaggerated ovarian androgen production early in the course of polycystic ovary syndrome.

Excess ovarian androgen production might be a cause of the polycystic ovary syndrome (PCO). Previous studies have evaluated adult women with long-standing abnormality of the hypothalamic-pituitary gonadal axis. Abnormal ovarian function in such patients could be a primary or even a secondary finding. For that reason, this study was designed to evaluate ovarian androgen production in symptomatic adolescent females. Simultaneous adrenal suppression, by using dexamethasone, and ovarian stimulation, by using gonadotropin-releasing hormone (GnRH), were achieved in 12 patients. Following stimulation, blood was serially obtained over 8 hr to measure gonadotropin, estrogen, and androgen responses. Based on the androgen response, patients could be divided into two groups. Group A (five) had a significant increase (p less than 0.01) in free testosterone, whereas group B (seven) had no increase in any androgen, including free testosterone (significantly different from group A, p = 0.01). All patients in group A had enlarged or cystic ovaries, whereas only one-quarter patients in group B had enlarged ovaries (significantly different from group A, p less than 0.03). The pituitary and estrogenic response was similar in both groups. These preliminary data suggest that some patients with PCO (group A) have a primary abnormality in ovarian androgen production early in the course of their disease.

A relationship between neonatal breast size and cord blood testosterone level.

The present study was performed to determine if any hormone measured in cord blood correlates with the size of the neonatal breast or the presence of galactorrhea. A total of 144 term newborn infants were examined. Estradiol (E2), progesterone (P), testosterone (T), and thyrotropin (TSH) were determined by radioimmunoassay (RIA), and prolactin (PRL) was determined by both RIA and biological activity (BA). The female breast (8.5 +/- 2.0 mm) was found to be larger than that of the male (7.8 +/- 2.1 mm, p less than 0.05). The only hormonal difference between sexes was a higher T level in the male infants (8.0 +/- 3.0 nmol per L vs. 5.5 +/- 1.9 nmol per L, p = 0.002). None of the other hormones measured by RIA correlated with the size of the neonatal breast or the presence of galactorrhea. The BA of PRL was widely variable compared to the PRL RIA but also failed to correlate with neonatal breast size or galactorrhea. This study suggests that T might be one factor in determining the size of the neonatal breast.

The effect of nicotine on the attachment of human fibroblasts to glass and human root surfaces in vitro.

This study examined the effect of nicotine on fibroblast attachment to glass and nondiseased human root surfaces. Human foreskin fibroblasts (HFF) were trypsinized, suspended in RPMI 1640 medium, and incubated with autoclaved human root sections and nicotine concentrations of zero (control), 25, 50, 100, 200, or 400 ng/ml. The root sections were examined for fibroblast attachment at 24, 48, and 72 hours by light microscopy and scanning electron microscopy (SEM). Additional trypsinized HFF were incubated on glass surfaces with the same concentrations of nicotine and examined at one week by light microscopy. HFF attached and grew on glass and root surfaces at all concentrations of nicotine. Controls on glass surfaces exhibited a normal monolayer of long spindle-shaped fibroblasts with a parallel alignment and minimal overlapping. Nicotine-treated HFF exhibited a haphazard arrangement with cell overlapping and vacuolization of the cytoplasm. Under SEM, the controls had smooth surfaces and appeared firmly attached to the root surface via (1) microvilli and filopodia on the cell boundaries and (2) short, branched, thin-to-medium width cytoplasmic processes with microvilli and filopodia on their boundaries. Few microvilli were noted on the control cell surfaces. HFF exposed to nicotine had microvilli and filopodia on the cell surfaces and long thin and long broad cytoplasmic processes with many microvilli and filopodia that projected away from the root surface. These findings suggest that the nature of fibroblast attachment to glass and root surfaces is altered by nicotine. A similar disturbance in fibroblast attachment may occur in humans who use nicotine-containing products, making them more susceptible to destruction of the periodontium and less responsive to new attachment after periodontal therapy.

Expression of Agrobacterium tumefaciens T-DNA gene 7 in Xenopus laevis oocytes.

The expression of Agrobacterium tumefaciens T-DNA gene 7 was investigated in Xenopus laevis oocytes. Cloned DNA injected into oocytes consisted of T-DNA sequences derived from octopine type Ti plasmid B6-806 and T-DNA attached to plant DNA sequences at the left junction in crown gall tumors. Transcription initiation sites observed in oocytes were similar to those for transcript 7 in crown gall tumors. Quantitative differences in transcription occurred depending on the flanking sequences of the injected clones indicating that sequences upstream of the TATA box of T-DNA gene 7 affect the quantitative expression of this gene in Xenopus oocytes.

A simple method for determination of red blood cell mechanical fragility in the rat.

A method for measuring the mechanical fragility of red blood cells suitable for use in small laboratory animals, such as rats, is reported because of lack of such data in the literature. Whole blood is mixed with phosphate buffered saline in a tube containing glass beads. The tubes are rocked for 90 minutes, centrifuged and the percent hemolysis determined. Varying the osmolality of the saline suspending medium had little effect on the mechanical fragility of rat red cells prior to the NaCl concentrations at which a significant change in osmotic hemolysis occurred. The duration of rocking increased the mechanical fragility. Varying the pH (6.4-8.0) had no effect. The size of the glass beads changed the mechanical fragility as did varying temperature. The mean mechanical fragility of rat red blood cells was 46% hemolysis (80 adult male animals). Because of the small volume of blood required with this method, mechanical fragility of red cells of other small laboratory animals also may be determined.