Callus Composition Research Articles

Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit ulnar osteotomy model

This study evaluated healing of rabbit bilateral ulnar osteotomies 6 and 8 weeks after surgery in response to percutaneous injection of transgenic adenoviral (Ad) bone morphogenetic protein-6 (BMP-6) vector or green fluorescent protein vector control (Ad-GFP) administered 7 days after surgery compared to untreated osteotomy controls. The amount, composition and biomechanical properties of the healing bone repair tissue were compared among groups and to historical data for intact rabbit ulnae obtained from similar studies at the same institution. Quantitative computed tomography was used to determine area, density and mineral content of the mineralized callus in the harvested ulnae. Maximum torque, torsional stiffness, and energy absorbed to failure were determined at 1.5°/s. Calcified sections of excised ulnae (5 μm) were stained with Goldner's Trichrome and Von Kossa, and evaluated for callus composition, maturity, cortical continuity, and osteotomy bridging. Radiographic assessment of bone formation indicated greater mineralized callus in the ulnae injected with Ad-hBMP-6 as early as 1 week after treatment (2 weeks after surgery) compared to untreated osteotomy ulnae ( p<0.006) and Ad-GFP treated osteotomy ulnae ( p<0.002). Quantitative computed tomography confirmed greater bone area and bone mineral content at the osteotomy at 6 weeks in Ad-BMP-6 treated osteotomy as compared to untreated osteotomy ulnae ( p<0.001) and Ad-GFP treated osteotomy ulnae ( p<0.01). Ad-BMP-6 treated osteotomy ulnae were stronger ( p<0.001 and 0.003) and stiffer ( p=0.004 and 0.003) in torsion at 6 weeks than untreated osteotomy ulnae or Ad-GFP treated osteotomy ulnae, respectively. Maximum torque, torsional stiffness, and energy absorbed to failure were greater in Ad-BMP-6 treated osteotomy ulnae compared to their respective untreated contralateral osteotomy ulnae at 8 weeks [ p<0.03]. Maximum torque and torsional stiffness in the Ad-BMP-6 treated osteotomy ulnae were not different to intact ulnae values at 6 and 8 weeks. These experiments confirm that BMP-6 can be potently osteoinductive in vivo resulting in acceleration of bone repair.

Long-term effects of local growth factor (IGF-I and TGF-$beta;1) treatment on fracture healing*1A safety study for using growth factors

Previous studies showed that growth factors dramatically stimulate healing processes in bone. However, the long-term effect of locally applied growth factors on fracture healing remains unclear. In considering the safety of using growth factors, it is necessary to elucidate that after initial stimulation, the effect stops and the result is a normally healed tissue. Therefore, the purpose of the present study was to investigate the long-term time course of healing processes during growth factor (GF) stimulated and unstimulated fracture healing in a closed tibial fracture model in rats. A well established local drug delivery system was used. IGF-I (50 μg) and TGF-β1 (10 μg) were locally applied using a 10 μm thin polylactide (PDLLA) coating on intramedullary implants. The biomechanical and histomorphometrical results demonstrated a significant stimulation of the fracture healing due to the locally applied growth factors compared to control at days 28 and 42 in agreement with the literature. At the last time point, 84 days after fracture, no differences were measurable in the biomechanical testing and the callus composition between the groups. The callus was consistently in the late phase of remodeling with no remaining cartilage. In conclusion, local growth factor application enhances the healing in the early phase without alteration of the normal healing process. © 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Local delivery of growth factors from coated titanium plates increases osteotomy healing in rats

Different methods for the stabilization of long bone fractures are used in clinic. Besides the development of further stabilization devices, the use of new materials, the modification of the surfaces, and the local application of stimulating factors for enhancement of healing are from great interest. Previous studies successfully used a biodegradable poly( d, l-lactide) coating as a local drug delivery system of growth factors from intramedullary (IM) implants to enhance fracture healing. In this study, we developed a new rat model ( n = 60) for plate osteosynthesis and used a plate for stabilization and as a local drug delivery system for the growth factors IGF-I and TGF-β1. A four-hole titanium plate was used for stabilization of a 0.6-mm osteotomy gap of the femur. The space between the inner holes was coated with 50 μg IGF-I and 10 μg TGF-β1 incorporated in the poly( d, l-lactide) coating or with the coating alone. After 42 days, biomechanical tests and histomorphological analyses were performed to investigate osteotomy healing. Radiologically small differences were detectable between the groups. The biomechanical torsional testing revealed a significantly higher maximum load of the osteotomized femura after treatment with growth factors compared to the uncoated group. In the histomorphometric analyses measuring the callus composition, a significantly higher percentage of mineralized tissue in the osteotomy callus was assessed in the growth factor treated group compared to the uncoated. In conclusion, the local application of IGF-I and TGF-β1 from a biodegradable coating enhances the osteotomy healing as shown in the biomechanical testing and the histomorphometry. Bioactive plates could be used in clinic for fracture stabilization and for local and controlled application of growth factors to stimulate bone healing.

Long-term effects of local growth factor (IGF-I and TGF-β1) treatment on fracture healing: A safety study for using growth factors

Previous studies showed that growth factors dramatically stimulate healing processes in bone. However, the long-term effect of locally applied growth factors on fracture healing remains unclear. In considering the safety of using growth factors, it is necessary to elucidate that after initial stimulation, the effect stops and the result is a normally healed tissue. Therefore, the purpose of the present study was to investigate the long-term time course of healing processes during growth factor (GF) stimulated and unstimulated fracture healing in a closed tibial fracture model in rats. A well established local drug delivery system was used. IGF-I (50 μg) and TGF-β1 (10 μg) were locally applied using a 10 μm thin polylactide (PDLLA) coating on intramedullary implants. The biomechanical and histomorphometrical results demonstrated a significant stimulation of the fracture healing due to the locally applied growth factors compared to control at days 28 and 42 in agreement with the literature. At the last time point, 84 days after fracture, no differences were measurable in the biomechanical testing and the callus composition between the groups. The callus was consistently in the late phase of remodeling with no remaining cartilage. In conclusion, local growth factor application enhances the healing in the early phase without alteration of the normal healing process.

Improvement of fracture healing by systemic administration of growth hormone and local application of insulin-like growth factor-1 and transforming growth factor-β1

Fracture healing is influenced by numerous hormones, growth factors, and cytokines. The systemic administration of growth hormone (GH) has shown to accelerate bone regeneration. Local application of growth factors, such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-β-1 (TGF-β1), are known to stimulate bone metabolism. Until now, the exact local and systemic mechanisms that lead to improved bone regeneration remain unclear. In addition, the effect of systemic administration of GH as compared with locally delivered growth factors on fracture healing in rats is not known. A midshaft fracture of the right tibia of 5-month-old female Sprague-Dawley rats (n = 80) was intramedullary stabilized with IGF-1 and TGF-β1 coated vs. uncoated titanium K-wires. The growth factors were incorporated in a poly(D,L-lactide) (PDLLA) coating and released continuously throughout the experiment. Recombinant species-specific (rat) GH was applied systemically (2 mg/kg body weight) by daily subcutaneous injection and compared with a placebo group. The healing process was radiologically monitored. Twenty-eight days after fracture biomechanical torsional testing was performed. The consolidation and callus composition, including quantification of cartilage and mineralized tissue, was traced in histomorphometrical investigations using an image analysis system. Both methods, the systemic administration of GH and the local application of growth factors, showed significant biomechanical and histological effects on fracture healing. The local growth factor application showed a stronger effect on fracture healing than the systemic GH injection. The combined application of both methods did not accelerate the effect on bone healing compared with the single application. It is therefore concluded that combining local and systemic stimulating methods does not provide further additive effects with regard to fracture healing.

Volatile constituents of callus and flower-bearing tops ofZataria multiflora Boiss. (Lamiaceae)

Green callus of Zataria multiflora Boiss. (Lamiaceae) was induced from seedlings. The detailed volatile composition of callus and of flower-bearing tops used in the callus induction was investigated by GC and GC – MS spectrometry. Callus volatile constituents consisted of monoterpenoids (25.59% monoterpene hydrocarbons and 70.99% oxygenated monoterpenes), while volatile oil of the flowering tops consisted mainly of monoterpenoids (20.95% monoterpene hydrocarbons and 73.07% oxygenated monoterpenes) and 4.62% sesquiterpene hydrocarbons. The typical components for the two volatile oils were p-cymene (4.85% and 5.43%), γ-terpinene (19.95% and 7.74%), thymol (34.91% and 32.35%) and carvacrol (5.06% and 25.95%). Furthermore, thymol acetate (22.60%) and carvacrol acetate (5.95%) were found mainly in the former and linalol (6.77%) was found merely in the latter. Copyright © 2000 John Wiley & Sons, Ltd.

Lipid changes due to growth-factor supplements in callus and plasma membrane-enriched fraction of rice cultures

Membrane-lipid composition of total callus and its plasma membrane enriched fraction of cultures of Oryza sativa grown on media supplemented with various growth factors, such as choline, putrescine and pectin, was determined. Whereas choline was growth stimulatory, putrescine and pectin inhibited growth in these cultures. Growth response due to the growth-factor supplements was associated with significant changes in the total content, as well as the relative amounts of phospholipids and galactolipids. Whereas choline supplementation led to an increase in the relative amounts of phosphatidylcholine and phosphatidylinositol, putrescine/pectin supplemented cultures showed higher level of phosphatidic acid. A plasma membrane-enriched fraction of choline-supplemented cultures had higher content of phospholipids and galactolipids, together with enriched contents of phosphatidyl-choline. Results are discussed in relation to the role of membrane lipids in in vitro growth.

In vitro culture of coconut endosperm: callus induction and its fatty acids

Successful induction of callus from coconut endosperm was achieved by using the tissue situated near the micropylar end of a young fruit. For initiation of callus, a high concentration of auxin (20 to 100 ppm) was added to the basal medium containing activated charcoal. Subcultured callus showed a 40-fold increase during culture of three months. Based on the analysis of fatty acid composition, the maturation of endosperm was characterized by an increase in short chain fatty acids (C8, C10, C12, C14)and a decrease in long chain fatty acids (C16, C18: 1, C18: 2). In developing endosperms, proportion of short chain fatty acids was higher in lipids of the antipodal than those of other regions. In the final stage of maturation, around 82% of total fatty acids was short chain fatty acids, while the proportion of long chain fatty acids decreased up to 16%. The fatty acid composition of callus subcultured for six months was comparable to that of the immature endosperm. Lipids were accumulated in callus as globular bodies.

Changes in fatty acid and lipid composition in normal and habituated sugar beet calli

Analysis of fatty acids and other classes of lipids in normal and habituated calli of Beta vulgaris shows that their lipid composition is identical during early stages of culture. However, after 14 days this composition turns out to be very different, as a result of membrane structure modifications during the growth of both types of calli. The fatty acid composition of normal callus remains very stable. The loss of phospholipids during culture is compensated for by increased sterol contents. The normal callus, therefore, seems to maintain its membrane integrity throughout growth. On the contrary, the abundance of free fatty acids and malondialdehyde in habituated callus, even at the juvenile stage, together with a decrease of the unsaturation level of fatty acids during culture are indicative of high lipid peroxidation. Peroxidation of polyunsaturated fatty acids and increases in neutral lipids could generate a loss of fluidity in membranes of habituated callus; the more permeable calli would be unable to maintain their homeostasis. This disturbed cellular homeostasis could be correlated with the vitrified aspect of the habituated callus and its relation with the acquisition of hormonal autonomy could be questioned.

Senescence-related lipid changes in callus cultures of Datura innoxia

Lipid changes related to abscisic acid (ABA) induced senescence was studied in callus cultures of Datura innoxia. Cultures attained terminal senescence in four weeks due to supplementation of ABA. Senescence was marked by loss of chlorophyll, decrease in protein content and profound changes in lipid composition. Neutral lipids constituted major fraction of cellular lipids together with complete degradation of galactolipids of the senesced callus. In contrast to the phospholipid composition of healthy callus which consisted of phosphatidylcholine (PC) -ethanolamine, -inositol, -serine, -glycerol and phosphatidic acid (PA), the senesced callus had only PE and PC along with their lysoderivatives and PA. The results are discussed in relation to ABA-induced senescence and significance of changes in membrane lipid composition.

Preliminary observation of the chemical composition of callus derived from immature seeds ofVernonia galamensis, Var. Ethiopica, Gilbert

Preliminary observation of the chemical composition of callus derived from immature seeds of<i>Vernonia galamensis</i>, Var. Ethiopica, Gilbert

Cocobean Tissue Culture: Lipid Composition and Fatty Acid Metabolism

ABSTRACTCocoabean callus cultures were established and grew satisfactorily on an agar medium for more than 2 yr. Cell suspension cultures were initiated from the callus. The fresh weight of cells increased over 20‐fold in 14 days. The lipid content of callus and cells was 5.3 and 6.5%, respectively. The fatty acid composition of cocoa callus and cell suspension cultures resembled that of immature cocoabeans since they contained high amounts of linoleic acid. Stereospecific distribution of fatty acids in triglycerides from cocoa callus and cells was similar to that of ripe cocoabeans. Exogenous acetate and fatty acids were readily incorporated into lipids by cocoa cell suspension cultures. Exogenous stearic acid increased triglyceride content twofold but did not change fatty acid composition of triglycerides. Coconut water alone or in combination with sucrose also increased triglyceride content with a concomitant increase in oleic acid from 10% to 33% and a decrease in linoleic acid from 37% to 19%.

Effect of ibuprofen on mass and composition of fracture callus and bone. An experimental study on adult rat.

Sixty adult male rats were used to study the effect of the anti-inflammatory drug Ibuprofen on fracture callus and perpendicular skeleton. After experimental periods of 3 and 9 weeks, fracture callus and both fractured and unfractured tibiae were examined with respect to bone mass and composition and 45Ca metabolism. No significant changes were found in the composition of fracture callus during treatment. Significantly diminished parameters of both fractured and unfractured tibia were observed for wet and dry weights, ash content, and organic matter after 3 weeks but the bone mass had become almost restored and the changes were non-significant during treatment 9 weeks following fracturing. The 45Ca activity was elevated significantly in fracture callus and fractured tibia 3 weeks after fracturing but had definitely declined to physiological levels at 9 weeks. Serum 45Ca activity was significantly elevated during Ibuprofen treatment. The findings support the concept that Ibuprofen lessens the bone mass and composition of both fractured and unfractured tibia and also activates the calcium metabolism in fracture callus. In the long run, however, this effect is weakened and the bone changes are become almost normal. Some explanations regarding these short-term effects of Ibuprofen are discussed.

Kinetin induced changes in cell wall composition of tobacco callus

Culture of tobacco callus on high or low kinetin in light or darkness leads to changed tissue texture and associated changes in cell wall composition. In particular, friable callus (low kinetin, darkness) cell walls have a greater extensin content and an altered composition of arabinose and xylose containing hemicelluloses compared with cell walls of compact callus (high kinetin, darkness). The possible importance of these differences in determining callus friability is discussed.

RNA Profiles and Nucleotide Composition of Flax Callus and Suspension Cultures

Absorbance profiles and nucleotide composition of RNA isolated from flax callus and cell suspension cultures were compared with those of intact cotyledons. The acrylamide gel electrophoretic patterns of total RNA from green callus and suspension cultures were characterized by the absence of both the chloroplast ribosomal RNA species, 23 S and 16 S. Furthermore, the nucleotide composition of total and individual RNA fractions of both cultures exhibited a high content of UMP and a corresponding decrease in CMP as compared with those of the cotyledons. The CMP/AMP ratio, however, was higher for the intact than for the cultured tissues. These changes were discussed in relation to the loss of biosynthetic potential for flavonoid compounds by the cultured tissues.