Osteoinductivity of demineralized bone matrix in immunocompromised mice and rats is decreased by ovariectomy and restored by estrogen replacement.

The osteoinduction potential of human demineralized bone matrix (DBM) in females with low estrogen (E2) is unknown. Moreover, the osteoinductivity of commercial human DBM is tested in male athymic rats and mice, but DBM performance in these animals may not reflect performance in female animals or provide information on E2's role in the process. To gain insight, human DBM was implanted bilaterally in the gastrocnemius of twenty-four athymic female mice (10 mg/implant) and twenty-four athymic female rats (15 mg/implant). Eight animals in each group were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized with E2-replacement (OVX+E2) via subcutaneous slow release capsules of 17beta-estradiol. OVX and OVX+E2 animals were pair-fed to SHAM animals. Four animals from each group were euthanized at 35 days and four at 56 days. Animal weight, uterine weight, and blood estrogen levels confirmed that pair feeding, ovariectomy, and E2 replacement were successful. Histological sections of implanted tissues were evaluated qualitatively for absence or presence of DBM, ossicle formation, and new bone or cartilage using a previously developed qualitative scoring system (QS) and by histomorphometry to obtain a quantitative assessment of osteoinduction. OVX mice had a small but significant QS decrease at 35 days compared to SHAM mice, confirmed by quantitative measurement of ossicle, marrow space, and new bone areas. The QS in rats was not affected by OVX but histomorphometry showed decreased new bone in OVX rats, which was restored by E2. The QS indicated that the number of new bone sites was not reduced by OVX in rats or mice at 56 days, but the relative amount of new bone v. marrow space was affected and differed with animal species. Residual DBM was less in OVX animals, indicating that DBM resorption was affected. Cartilage was present in rats but not in mice, suggesting that endochondral ossification was slower and indicating that bone graft studies in these species are not necessarily comparable. These results show the importance of E2 in human DBM-induced bone formation and suggest that E2 may be needed for clinical effectiveness in post-menopausal women.

Human articular chondrocytes exhibit sexual dimorphism in their responses to 17beta-estradiol.

OBJECTIVE: The higher incidence of osteoarthritis in females suggests that there may be intrinsic sex-specific differences in human articular chondrocytes. 17beta-Estradiol (E2) regulates rat growth plate chondrocytes through traditional nuclear receptor mechanisms, but only female cells exhibit rapid membrane-associated effects mediated through protein kinase C (PKC) alpha. Here we demonstrate sexual dimorphism in the physiological response of human articular chondrocytes to E2. METHODS: Articular chondrocytes were obtained at the time of autopsy from three male and three female donors between 16 and 39 years of age. Second passage cultures were treated with E2 for 24 h to assess the effects of the hormone on [3H]-thymidine incorporation, [35S]-sulfate incorporation, and alkaline phosphatase specific activity. In addition, the chondrocytes were treated for 3, 9, 90 or 270 min and PKC specific activity was determined. RESULTS: All chondrocytes were positive for aggrecan and estrogen receptor alpha mRNAs but were negative for type II collagen mRNA. Only cells from female donors responded to E2. DNA synthesis, sulfate incorporation and alkaline phosphatase activity were increased. E2 caused a rapid increase in PKC activity in the female cells within 9 min that was maximal at 90 min. Treatment with the PKC inhibitor chelerythrine blocked these effects. CONCLUSIONS: These results provide the first definitive evidence that normal human cells exhibit an intrinsic sex-specific response to E2 and suggest that sexual dimorphism may be an important variable in assessing the pathways that modulate cell behavior.

Sciatic nerve blockade with lipid-protein-sugar particles containing bupivacaine.

PURPOSE: To assess the efficacy of lipid-protein-sugar particles (LPSPs) in providing prolonged duration local anesthesia by percutaneous injection. METHODS: Bupivacaine-containing LPSPs were characterized and optimized in vitro. Male Sprague-Dawley rats were given sciatic nerve blocks with bupivacaine-containing LPSPs. Sensory and motor nerve blockade were measured in the hindpaw, as were contralateral functional deficits (a measure of systemic drug distribution). Poly(lactic-co-glycolic) acid (PLGA) microspheres were used as a reference. RESULTS: 10% (w/w) bupivacaine LPSPs (60% dipalmitoylphosphatidylcholine) were 4.4+/-0.39 microm in diameter, with a tap density of 0.11 +/-0.04 g/ml. These LPSPs and 50% (w/w) PLGA microspheres had comparable durations of sensory blockade (468+/-210 min vs. 706+/-344 min, p = 0.08), although the LPSPs produced a much lesser duration of motor blockade (508+/-258 min vs. 1062+/-456 min, p = 0.005). Systemic toxicity was minimal in both groups. CONCLUSIONS: LPSPs provide sensory blockade durations comparable to those from PLGA microspheres, with a smaller amount of drug loading. Motor blockade is shorter with LPSPs than with PLGA microspheres. LPSPs appear to be suitable for extended nerve blockade. Given their size and low density, they may be useful for topical anesthesia of the airway.

Intrathecal dynorphin A1-13 and dynorphin A3-13 reduce rat spinal cord blood flow by non-opioid mechanisms.

Radiolabeled microspheres were used to examine the effects of paralytic intrathecal doses of dynorphin A (Dyn A1-13) and Dyn A3-13 on rat brain and spinal cord blood flows and cardiac output. Dyn A1-13 produced significant dose-related reductions in blood flow to lumbosacral and thoracic spinal cord without altering cardiac output and blood flow to brain and cervical spinal cord. Naloxone failed to block these effects. Dyn A3-13, which lacks opioid activity, also significantly reduced blood flow in lumbosacral spinal cord. Thus, the paralytic effects of Dyn A in the rat may involve reductions in spinal cord resulting from non-opioid actions of Dyn A.

A laboratory model for studying blast overpressure injury.

Blast injury remains an important source of trauma in both civilian and military settings. We have studied a recently developed blast wave generator to evaluate its effectiveness for laboratory study of blast injury. In order to determine the reliability of the device and the pathology of the lesions caused by the short duration (0.5-1.0 msec), and high intensity (60-375 psi) pressure wave, laboratory rats were exposed to the pressure waves generated by the machine. The animals were divided into three groups: the first exposed to midthoracic blasts, the second to abdominal blasts, and a group of controls exposed to a gentle stream of gas. Group I showed gross and microscopic evidence of lung blast injury of "rib imprint" hemorrhages, intra-alveolar hemorrhage, marked increase in lung weight, prolonged apnea, and bradycardia. Group II showed typical blunt abdominal trauma at the closest ranges, but characteristic submucosal hemorrhages up to 4.0 cm from the blast nozzle. In both groups, a protective effect was seen in heavier animals. The blast wave generator permits reproducible blast injury in the laboratory that is safer and faster than current methods. The lung and bowel lesions induced are grossly and microscopically similar to injuries of blast exposure seen in clinical patients.

Intrathecal dynorphin A (1-13) and (3-13) reduce spinal cord blood flow by non-opioid mechanisms.

Dynorphin A (Dyn A)-related peptides have been implicated in the pathophysiology of spinal cord injury in part because their intrathecal (i.t.) injection causes hindlimb paralysis. The effects of paralytic doses of i.t. Dyn A (1-13) and Dyn A (3-13) on spinal cord blood flow and cardiac output were examined in rats using radiolabeled microspheres. Both Dyn A (1-13) and Dyn A (3-13) significantly reduced blood flow in lumbosacral spinal cord without altering cardiac output. Pretreatment with naloxone failed to block these reductions in blood flow. Thus, the paralytic effects of Dyn A may result from non-opioid actions of Dyn A to reduce spinal cord perfusion.