Bone Morphogenetic Protein 7 (BMP-7) Influences Tendon-Bone Integration In Vitro

IntroductionSuccessful graft ingrowth following reconstruction of the anterior cruciate ligament is governed by complex biological processes at the tendon-bone interface. The aim of this study was to investigate in an in vitro study the effects of bone morphogenetic protein 7 (BMP-7) on tendon-bone integration.ResultsIn both models, positive effects of BMP-7 on ALP enzyme activity were observed (p<0.001). Additionally, similar results were noted for LDH activity and lactate concentration. BMP-7 stimulation led to a significant increase in OCN expression. Whereas the effects of BMP-7 on tendon monoculture peaked during an early phase of the experiment (p<0.001), the cocultures showed a maximal increase during the later stages (p<0.001). The histological analysis showed a stimulating effect of BMP-7 on extracellular matrix formation. Organized ossification zones and calcium carbonate-like structures were only observed in the BMP-stimulated cell cultures.DiscussionThis study showed the positive effects of BMP-7 on the biological process of tendon-bone integration in vitro. Histological signs of improved mineralization were paralleled by increased rates of osteoblast-specific protein levels in primary bovine osteoblasts and fibroblasts.ConclusionOur findings indicated a role for BMP-7 as an adjuvant therapeutic agent in the treatment of ligamentous injuries, and they emphasized the importance of the transdifferentiation process of tendinous fibroblasts at the tendon-bone interface.     

Simple reversed-phase high-performance liquid chromatography quantitation of ganciclovir in human serum and urine

A fast, simple, and cost-effective HPLC method for the quantitation of the antiviral drug ganciclovir is described. The serum samples are extracted with perchloric acid and neutralized with potassium phosphate buffer, and urine samples are diluted with distilled water. A reversed-phase column with isocratic elution by 15 mM potassium phosphate buffer (pH 2.5) containing 0.25% acetonitrile is used to separate ganciclovir; quantitation is by UV absorbance at 254 nm. Total turnaround time is 22 min; more than 3000 samples can be run on a single column without loss of peak quality. The limit of quantitation is 0.05 μg/ml. Recoveries varied from 91 to 10% with coefficients of variation ranging from 0.387 to 7.95%.     

Inhibitory effects of HgCl2 on excitation-secretion coupling at the motor nerve terminal and excitation-contraction coupling in the muscle cell

Summary 1. Indirect and direct twitch (0.1-Hz) stimulation of the rat phrenic nerve-diaphragm disclosed that the inhibitory effect of HgCl₂, 3.7 × 10^–5 M, on the neuromuscular transmission and in the muscle cell, was accelerated by 10-sec periods of 50-Hz tetanic stimulation every 10 min. This activity-dependent enhancement suggested an inhibitory mechanism of HgCl₂ related to the development of fatigue, like membrane depolarization or decreased excitability, decreased availability of transmitter, or interference with the factors controlling excitation-secretion coupling of the nerve terminal, i.e. (Ca²⁺)₀ or (Ca²⁺)_i, and excitation-contraction coupling in the muscle cell, i.e., (Ca²⁺)_i.2. During both indirect and direct stimulation, HgCl₂-induced inhibition was enhanced markedly by pretreatment with caffeine, which releases Ca²⁺ from endoplasmic and sarcoplasmic reticulum in the nerve terminal and muscle cell, respectively. This caffeine-induced enhancement was completely antagonized by dantrolene, which inhibits the caffeine-induced release. However, dantrolene alone did not antagonize the HgCl₂-induced inhibition.3. Since caffeine depletes the intracellular Ca²⁺ stores of the smooth endoplasmic reticulum, HgCl₂ probably inhibits by binding to SH groups of transport proteins conveying the messenger function of (Ca²⁺)_i. In the muscle cell this leads to inhibition of contraction. In the nerve terminal, an additional enhancement of the HgCl₂-induced inhibition, by inhibiting reuptake of choline by TEA and tetanic stimulation, suggested that HgCl₂ inhibited a (Ca²⁺)_i signal necessary for this limiting factor in resynthesis of acetylcholine.4. The (Ca²⁺)₀ signal necessary for stimulus-induced release of acetylcholine was not affected by HgCl₂. Hyperpolarization in K⁺-free solution antagonized the inhibitory effect of HgCl₂ at indirect stimulation, and Ca²⁺-free solution enhanced the inhibitory effect at direct stimulation. K⁺ depolarization, membrane electric field increase with high Ca²⁺, membrane stabilization with lidocaine, and half-threshold stimulation, did not change the inhibitory effect of HgCl CH₃HgCl, 1.85 × 10^–5 M, disclosed a synergistic interaction with caffeine during direct, but not during indirect, stimulation.     

Pleiotropic action of the murine quaking locus: Structure of the qk v allele

The spontaneous allele quaking^viable (qk ^v ) exerts effects on myelination and spermiogenesis. The defects generated by qk ^v were not separated in a multilocus mapping cross that provided a mapping resolution of 0.1 centiMorgans (cM). Furthermore, no distortions suggestive of a large chromosomal anomaly associated with qk ^v were apparent. One plausible interpretation is that the quaking locus contains more than one functional domain, either organized into overlapping genes or expressed by alternative splicing mechanisms. The cloning needed to analyze this locus will be enhanced by the very high resolution of the meiotic mapping cross reported here. The recombinational distances on this qk ^v map were compressed compared with those previously reported in a high-resolution map for qk ^1–1, an embryonic lethal allele of quaking induced by ethylnitrosourea. Additional crosses confirmed prior reports that the sex and the genetic background of the heterozygous parent can affect recombinational distances. These joint effects on recombination are strong enough to account for the discrepancy between the two maps. This variability of two-factor map values leads to the preferred multilocus map-building protocol discussed in the accompanying paper.     

Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat

Summary Two previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.     

Interstitial bone stress distributions accompanying ingrowth of a screen-like prosthesis anchorage layer

Recent development of screen-like bonded weaves of titanium wire for orthopaedic implant anchorage affords a unique opportunity for analytic studies of porous ingrowth micromechanics. The regular geometry of individual wires and the periodicity of the mesh weave are exploited in a series of two-dimensional finite element models, mapping interstitial bone stress fields as a function of ingrowth depth and wire size, shape, and spacing.

When the depth of bone ingrowth was less than one wire diameter, peak bone stresses always occurred at the leading (i.e. deepest) edge of bone ingrowth, immediately adjacent to the wire. As ingrowth depth approached a full wire diameter, peak local bone stresses were 2–9 times the nominal applied host bone stress, with greater stresses occurring for lower screen weave densities. Within multiple screen layers, the top layer consistently experienced the peak stress and transmitted most of the applied load, regardless of the number of underlying screen layers surrounded by bone. Neither wire size variations nor partial wire flattening substantially affected general trends in stress predictions.