Demineralized bone matrix as a biological scaffold for bone repair.

Experimental models were created in rat fibula to represent impaired bone healing so that biological deficiencies that cause bone repair to fail or to be delayed may be investigated. These models consist of a 4-mm-long segmental defect, created in rat fibula by osteotomy, and fitted with a 7-mm-long tubular specimen of demineralized bone matrix (DBM) over the cut ends of the fibula. The experiments in this study involved various modifications of the DBM scaffold designed to reduce its osteoinductive activity: steam sterilization (sDBM), ethylene oxide sterilization (eoDBM), trypsin digestion (tDBM), and guanidine hydrochloride extraction (gDBM). Bone healing was evaluated by bending rigidity of the fibula and mineral content of the repair site at 7 weeks post-surgery. The sDBM scaffolds resorbed completely by 7 weeks and hence this model was a nonhealing negative control. Rigidities in the unmodified DBM and tDBM groups were comparable, whereas in the gDBM and eoDBM groups it was significantly reduced. Histologically, in the 4-mm defects repaired with unmodified DBM, direct and endochondral bone formation in the scaffold and the defect resulted in a neocortex consisting of woven and lamellar bone uniting the broken bone by 7 weeks post-surgery. We conclude that the eoDBM and gDBM groups represent failure or delay of the bone repair process when compared with the unmodified DBM group in which the process is analogous to normal bone healing.

Repair of segmental bone defects in the rat: an experimental model of human fracture healing.

Bone repair models in animals may be considered relevant to human fracture healing to the extent that the sequence of events in the repair process in the model reflect the human fracture healing sequence. In the present study, the relevance of a recently developed segmental defect model in rat fibula to human fracture healing was investigated by evaluating temporal progression of rigidity of the fibula, mineral content of the repair site, and histological changes. In this model, a surgically created 2-mm-long defect was grafted with a 5-mm-long tubular specimen of demineralized bone matrix (DBM) by inserting it over the cut ends of the fibula. The temporal increase in rigidity of the healing fibula demonstrated a pattern similar to biomechanical healing curves measured in human fracture healing. This pattern was characterized by a short phase of rapidly rising rigidity during weeks 4-7 after surgery, associated with a sharp increase in the mineral content of the repair tissue. This was preceded by a phase of nearly zero rigidity and followed by a phase of slow rate of increase approaching a plateau. Histologically, chondroblastic and osteoblastic blastema originating from extraskeletal and subperiosteal (near fibula-graft junction) regions, infiltrated the DBM graft during the first 2 weeks. The DBM graft assumed the role of a "bridging callus." By weeks 6-8, most of the DBM was converted to new woven and trabecular bone with maximal osteoblastic activity and minimal endochondral ossification. Medullary callus formation started with direct new bone formation adjacent to the cortical and endosteal surfaces in the defect and undifferentiated cells in the center of the defect at 3 weeks. The usual bone repair process in rodents was altered by the presence of the DBM graft to recapitulate the sequential stages of human fracture healing, including the formation of a medullary callus, union with woven and lamellar bone, and recreation of the medullary canal.

Magnetic field induced inhibition of human osteosarcoma cells treated with adriamycin.

Morbidity resulting from the toxicity of chemotherapeutic drugs suggests that novel approaches are worthy of investigation. Development of the use of low intensity magnetic fields as an adjuvant to current treatment regimens to prevent metastatic disease may prove to be efficacious. Using a cell culture model, we have developed a magnetic field (MF) treatment that offers the possibility of lowering the therapeutic dose of these drugs and thereby reducing morbidity. Our studies have found that a low intensity (approximately 2 gauss) MF signal and a relatively low dose (0.1 microg/ml) of Adriamycin (ADR) inhibited proliferation of human osteosarcoma cells by 82%, whereas the MF and ADR acting individually caused only 19% and 44% inhibition, respectively.

Teratogenic effects of ethanol during hyperplastic growth in cardiac myocyte cultures.

Ethanol alters cellular growth and maturation, teratologic factors that are recognized as contributing to abnormal phenotypic expression. Cultured neonatal Sprague-Dawley rat cardiac myocytes were utilized to determine how ethanol alters growth and development. Two ethanol exposure paradigms were studied: (1) constant, to cultures in closed chambers for 7 days at low (10 mM) and high (50 mM) concentrations; and (2) periodic (24-hr) to cells during hyperplastic growth. In constantly exposed cultures, 10 and 50 mM ethanol concentrations depressed the rate of leucine incorporation and the rate of thymidine uptake during early hyperplastic growth (log phase growth). A resultant slower expansion of cell populations was noted. Although the period of maximum vulnerability appeared to be the hyperplastic growth phase, a second set of experiments using 10 and 50 mM ethanol were performed to assess the effects of short (24-hr) exposures. DNA synthesis was depressed during early hyperplastic growth compared with controls (days 2-4), reflected as a decrease in thymidine incorporation and smaller cell population. This study demonstrates that ethanol depresses both DNA and protein synthesis during hyperplastic growth resulting in an insufficient, protein-deficient cell mass, incapable of participating in normal embryogenesis.

Ethanol-induced teratogenic alterations in developing cardiomyocytes in culture.

The development of an in vitro cardiogenesis model was designed to enhance our understanding of the mechanism(s) of ethanol teratogenicity. Growth and development events in the model are similar to in vivo events. Time-specific and event-specific windows of biokinetic activities during both hyperplastic and hypertrophic growth are easily controlled and independently investigated. Systematic study of the effects of ethanol on the embryogenesis model, from committed blast cells to mature, functioning cells was accomplished to determine at what stage or stages of the growth and development paradigm ethanol exerts its teratogenic potential. Using the model and comparing the data to in vivo ethanol exposures, it appears that ethanol impairs the capability of the cell to properly propagate and mature.

Ethanol induced morphologic alterations during growth and maturation of cardiac myocytes.

Alteration of growth and development of cells exposed to ethanol during embryogenesis contributes to dysmorphism. The mechanism(s) of these alterations remains an enigma. This paper describes studies of an in vitro cardiac myocyte model in which the major effort was to investigate growth and development parameters in an obligate interacting multicellular system. The well defined events of in vitro myogenesis allow for documentation or dysgenesis and altered growth in the presence of the taratogen, ethanol. The cells exposed to ethanol did not mature morphologically or functionally compared with controls. Increasing concentrations of ethanol appear to have a graded damaging effect. The greater the concentration of ethanol the more profound the dyssynchronous growth. The morphologic correlates were multinucleation, and alteration in the ultrastructural organization of cell-cell contacts and myofilaments. Correlation of these findings with those observed in dysgenic muscle of human infants and rat pups exposed to ethanol in utero, may provide at least a partial understanding of the teratogenic manifestation of ethanol in embryogenesis and organogenesis.

Closed chamber system for delivery of ethanol to cell cultures.

The accuracy and consistency of the delivery of ethanol to cultured cells is important to determine effects on morphologic, biochemical and physiologic alterations. Open and closed chamber systems were evaluated to determine cytotoxic vs sublethal, potentially teratogenic effects on neonatal rat cardiac myocytes. The open system employed a variety of cell culture vessels. Cardiac cells were exposed directly to ethanol in the growth media at concentrations of 5-50 mM in Petri dishes, multiwell slides and multiwell chambers. Ethanol concentrations in the media in these open vessels decreased over 60% in a 24 hr incubation period. A closed system consisted of tightly sealed plastic containers in which the same vessels were used. The vessels were placed on a platform over a bath of ethanol-water. Cells were acclimated for 24 hr with ethanol in the bath at 200% of the final desired media concentration. Ethanol gradually diffused into the media to reach peak levels of 5, 10, 25 or 50 mM at 24 hr. After the 24 hr period, ethanol was added to both the media and bath at the desired concentration. Cells exposed gradually to ethanol in the closed chambers remained viable, but showed slower division and growth. A period of gradual acclimation is required to induce sublethal cellular effects rather than lethal effects. The diversity of cell systems and manipulations of cultures to study the potential teratogenic effects of ethanol are improved using such a closed chamber system.

Ethanol-induced cytoskeletal dysgenesis with dietary protein manipulations.

Ethanol-induced cytoskeletal abnormalities occur in many cell types, affecting structure and function of the respective organs. When affecting the developing myocyte, damage is responsible for the hypotonicity and congestive heart failure of Fetal Alcohol Syndrome (FAS) infants. To test the hypothesis that ethanol damages myocytes in utero, and determine that damage is not affected by dietary protein manipulations, female Sprague-Dawley rats were fed Leiber-DeCarli diets containing either 10% or 25% protein-derived calories, isocalorically balanced with or without 36% of the total calories containing ethanol. Muscle biopsies from rat pups of the ethanol-fed groups showed disorganized myocyte cytoskeleton: sarcomeric dysplasia with Z-band abnormality, actin in disarray, and granulofilamentous debris. Teratogenic effects of ethanol on myocytes occur in rat pups in utero, supporting the use of this model for studying ethanol effects on the developing cytoskeleton. We report ultrastructural evidence which provides a partial explanation of the mechanism of a well-known clinical phenomenon.