A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants

An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.     

Citrus tissue culture employing vegetative explants

Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth culture of shoots of C grandis without loss of regeneration capacity during 31 years, observed so far, are some other significant results. Plants of C. aurantifolia and C. sinensis raised from shoot meristem and micrografting were grown in a nethouse and those from nodal stem segments in the field along with the in vitro-raised plants of rootstocks, namely, C. jambhiri, C. karna and C. limonia. All the plants showed normal healthy growth. Significantly enough, the meristem regenerated plants of C. aurantifolia attained the reproductive phase just in 1 year of transplantation to soil similar to those raised from nodal stem segments of mature trees, which also produced normal fruits in the subsequent year while growing under field conditions. Thus, a significant fundamental concept of a maturity factor, carried over through as small a shoot meristem as 200 microm in length to cloned plants has been demonstrated. The concept is of far-reaching significance in citrus industry besides production of pathogen-free orchards.     

Full-thickness skin wound explants in tissue culture: a mechanical evaluation of healing.

This study was designed to evaluate biomechanically defined wound healing in full-thickness skin explants in tissue culture. The requirement for preculture incubation of wounds in situ was characterized. Full-thickness skin incisions were made in 44 rats and closed immediately. Wounds were incubated in situ for 0, 12, 24, 36, 48, 72, or 96 hours before harvesting and placement into tissue culture media for 6 weeks. Healing was evaluated by biomechanical criteria: tensiometric distraction to wound rupture generated true stress and energy absorption data. Burst-strength (maximum true stress) and toughness (energy absorption) were five times higher in the 48-hour group than in any other group; other groups were not different from each other. This study demonstrates long-term survival of full-thickness skin in culture and shows that full-thickness skin explants heal in tissue culture. Possible explanations for the narrow window of opportunity for harvest (48 hours, no more and no less) are discussed.     

Effects of mechanical stimulation induced by compression and medium perfusion on cardiac tissue engineering

Cardiac tissue engineering presents a challenge due to the complexity of the muscle tissue and the need for multiple signals to induce tissue regeneration in vitro. We investigated the effects of compression (1 Hz, 15% strain) combined with fluid shear stress (10^?2–10^?1 dynes/cm²) provided by medium perfusion on the outcome of cardiac tissue engineering. Neonatal rat cardiac cells were seeded in Arginine‐Glycine‐Aspartate (RGD)‐attached alginate scaffolds, and the constructs were cultivated in a compression bioreactor. A daily, short‐term (30 min) compression (i.e., “intermittent compression”) for 4 days induced the formation of cardiac tissue with typical striation, while in the continuously compressed constructs (i.e., “continuous compression”), the cells remained spherical. By Western blot, on day 4 the expression of the gap junction protein connexin 43 was significantly greater in the “intermittent compression” constructs and the cardiomyocyte markers (α‐actinin and N‐cadherin) showed a trend of better preservation compared to the noncompressed constructs. This regime of compression had no effect on the proliferation of nonmyocyte cells, which maintained low expression level of proliferating cell nuclear antigen. Elevated secretion levels of basic fibroblast growth factor and transforming growth factor‐β in the daily, intermittently compressed constructs likely attributed to tissue formation. Our study thus establishes the formation of an improved cardiac tissue in vitro, when induced by combined mechanical signals of compression and fluid shear stress provided by perfusion. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation

One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three-dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long-term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non-woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30-day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: (1) free-swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage.     

A versatile gel casting cum electrophoresis apparatus

A simple apparatus for vertical.,in situ, polyacrylamide or agarose gel casting as well as for the subsequent electrophoresis is described. The apparatus is completely leakproof and does not require any special device like clamps, O-rings, gaskets, grease etc. for sealing. Slab gels of various thickness (0.04 to 1.0 cm) can be made and the apparatus can be used for analytical or preparative purposes. Gel rods can also be cast and run in the device. Forward as well as reverse polarity electrophoresis of a sample can be run simultaneously in the apparatus. NCL Communication No.: 3077.     

A versatile low-cost apparatus for cell electrofusion and other electrophysiological treatments

Instrumentation is described which will generate AC fields and DC square pulses for cell electrofusion, electroporation and other electrophysiological experiments. The equipment can generate a constant or intermittent AC field and a single square pulse, either alone or in combination. It can also generate a train of intermittent square pulses. The components for the instrumentation are commercially available and can be assembled by persons with little or no training in electronics. A design is given for a mass treatment chamber that can be easily constructed and viewed with a microscope.     

A new versatile compression chamber for examination of living microorganisms

A new type of compression chamber for microscopic studies of living microorganisms is described. This broadly applicable instrument allows controlled slowing down, turning and compression of vagile organisms. It has been developed for use with different types of microscopes, especially the modern inverted versions. Due to the extremely flat design, illumination according to the Köhler principles is guaranteed. Therefore, all modern procedures such as bright-, darkfield, phase-contrast, differential interference contrast, polarized and fluorescent microscopy can be carried out. Once focused, parfocal objective lenses (even oil immersion high power objectives) can be interchanged without refocusing. The chamber is composed of three basic elements: (1) baseplate, with cylindrical head-piece, (2) compression ring for vertical adjustment, (3) rotor, non-threaded and gliding on viscous silicon. An upper and a lower circular coverslip is permanently glued to the rotor and baseplate respectively. Preparation and interchange of the specimens are rapidly done by lifting and subsequent insertion of the non-threaded rotor. Thus, the new chamber is an almost non-evaporating closed system and the organisms to be studied are preserved alive for many hours. Except when high power magnifications are used, the principles, on which the compression chamber is based, allow various modifications in outfitting and design, such as transformation into a continuous flow system for fresh- or seawater, or exchange of media at choice. The chamber and its function is compared with similar instruments, such as the rotocompressor and the Spoon microcompressor.     

Resistin release by human adipose tissue explants in primary culture

Resistin, also known as Fizz3 or ADSF, is a protein found in murine adipose tissue and inflammatory lung exudates. The present studies found that resistin was released by explants of human adipose tissue but the release was quite variable ranging from 3 to 158 ng/g over 48 h. The release of resistin was 250% greater by explants of omental than by explants of human subcutaneous abdominal adipose tissue. Resistin release by adipocytes was negligible as compared to that by the non-fat cells of adipose tissue. Leptin formation by adipocytes was 8-fold greater than its formation by the non-fat cells, while the formation of PAI-1 by adipocytes was 38% of that by the non-fat cells. The conversion of glucose to lactate as well as the formation of PGE(2) and IL-8 was approximately 15% of that by the non-fat cells. In contrast the release of IL-6 and IL-1beta by adipocytes was 4-7% of that by the non-fat cells while the formation of resistin and IL-10 by adipocytes was 2% of that by non-fat cells. The release of adiponectin by explants ranged from 1000 to 5000 ng/g over 48 h but did not correlate with that of resistin. The present data suggest that resistin release by explants of human adipose tissue in primary culture is largely derived from the non-fat cells present in the explants.     

Screening for strongly regenerative genotypes of spinach in tissue culture using subcultured root explants

A system for subculture of spinach (Spinacia oleracea L.) roots was established, and differences in regeneration; namely, embryogenic competence, among individuals of the `Nippon' cultivar were examined. Root tissues, excised from seedlings, were grown on medium without growth regulators and subcultured on the same medium and then on medium that contained 10 M naphthaleneacetic acid and 0.1 M gibberellic acid to induce callus formation. Calli were transferred to medium without growth regulators. All explants formed calli. However, the frequency of embryo formation varied among lines. Higher concentrations of gibberellic acid in the callus-induction medium had limited effects on somatic embryogenesis from poorly embryogenic lines. These results indicate that inherent factors are important for somatic embryogenesis in spinach and that the root subculture system is useful for identifying strongly regenerative genotypes among individuals of a single cultivar.     

Collenchyma: a versatile mechanical tissue with dynamic cell walls

Background

Collenchyma has remained in the shadow of commercially exploited mechanical tissues such as wood and fibres, and therefore has received little attention since it was first described. However, collenchyma is highly dynamic, especially compared with sclerenchyma. It is the main supporting tissue of growing organs with walls thickening during and after elongation. In older organs, collenchyma may become more rigid due to changes in cell wall composition or may undergo sclerification through lignification of newly deposited cell wall material. While much is known about the systematic and organographic distribution of collenchyma, there is rather less information regarding the molecular architecture and properties of its cell walls.

Scope and conclusions

This review summarizes several aspects that have not previously been extensively discussed including the origin of the term ‘collenchyma’ and the history of its typology. As the cell walls of collenchyma largely determine the dynamic characteristics of this tissue, I summarize the current state of knowledge regarding their structure and molecular composition. Unfortunately, to date, detailed studies specifically focusing on collenchyma cell walls have not been undertaken. However, generating a more detailed understanding of the structural and compositional modifications associated with the transition from plastic to elastic collenchyma cell wall properties is likely to provide significant insights into how specific configurations of cell wall polymers result in specific functional properties. This approach, focusing on architecture and functional properties, is likely to provide improved clarity on the controversial definition of collenchyma.     

A continuous-flow culture system for organ culture of large explants of adult tissue: Effect of oxygen tension on mouse molar periodontium

Summary A modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for pO₂, pCO₂ and pH during the culture period. Three-molar and singlemolar explants of periodontium were maintained for 48 hr in the CFCS at two different pO₂ ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [³H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that cell viability and incorporation of [³H]proline, probably into newly synthesized protein, increased with an increase in pO₂ and was related to a pO₂ gradient extending from the periphery to the center of the explants.     

A continuous-flow culture system for organ culture of large explants of adult tissue: effect of oxygen tension on mouse molar periodontium

A modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for pO2, pCO2 and pH during the culture period. Three-molar and single-molar explants of periodontium were maintained for 48 hr in the CFCS at two different pO2 ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [3H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that cell viability and incorporation of [3H]proline, probably into newly synthesized protein, increased with an increase in pO2 and was related to a pO2 gradient extending from the periphery to the center of the explants.