Gregory C. Saggers

Mast cells promote fibroblast populated collagen lattice contraction through gap junction intercellular communication

The release of mast cell granules is commonly associated with inflammation and fibrosis. However, does direct communication between mast cells and fibroblasts through gap junction intercellular communication (GJIC) occur? Fibroblast populated collagen lattice (FPCL) cast with mast cells show enhanced lattice contraction. Do released granules or GJIC between mast cells and fibroblasts promote enhanced lattice contraction? Mast cells preloaded with a fluorescent dye that readily passes through gap junctions were cast in FPCL. Dye passed from mast cells into fibroblasts within these cocultured mast cell‐FPCLs. Fatty acid amide hydrolase inhibitor blocks the breakdown of oleamide, which is a potent endogenous inhibitor of GJIC. GJIC was blocked for 3 days when mast cells were pulsed for 3 hours with fatty acid amide hydrolase inhibitor. Mast cells pretreated with fatty acid amide hydrolase inhibitor cast in cocultured mast cell‐FPCLs failed to enhance cocultured lattice contraction. Mast cell‐FPCLs made with mouse fibroblasts unable to generate GJIC failed to show enhanced lattice contraction. Degranulated mast cells were equal to intact mast cells at enhancing cocultured mast cell‐FPCL contraction. The supernatant from degranulated mast cells had no effect upon FPCL contraction. Therefore, enhanced mast cell‐FPCL contraction appears to be independent of mast cell granules, but dependent upon GJIC between fibroblasts and mast cells. We speculate that mast cell–fibroblast GJIC may play a role in fibrosis.

Stretching skin : undermining is more important than intraoperative expansion

The efficacy of intraoperative expansion in reducing the tension of wound closure was tested in young pigs. The young piglet as a model for studying human skin was characterized by finding a close similarity between the modulus of elastiticy of young piglet skin and human abdominoplasty and mammaplasty skin (range 12.8 to 23.7 N/mm2 for piglet skin, 14.3 to 19 N/mm2 for human skin). The tension required to close a standardized wound was determined before undermining, after undermining, and finally after intraoperative expansion. These measurements were performed in 10 young pigs with an average weight of 11.5 kg. Undermining the wound edges resulted in a significant decrease in the force required to close the wounds (p < 0.0001). Intraoperative expansion did not significantly decrease the tension. Previous work showing the importance of site and direction of pull on the tension for wound closure was confirmed in this study. Analysis of variance demonstrated that the tension required to close a standard wound is greater high on the pigs back than near the belly and near the shoulder as opposed to the hip for midflank wounds (p < 0.0001). Increasing the extent of undermining from 62 to 136 cm2 significantly decreased the tension for wound closure (p < 0.05). Further undermining did not result in a significant decrease in wound closure tension. In this model, intraoperative expansion offers no advantage over simple undermining. We suggest that the benefit reported by clinicians using intraoperative expansion may derive from an increase in the extent of undermining required to place expanders under the wound margins.

Aeromonas species isolated from medicinal leeches.

Aeromonas hydrophila infections are a recognized complication of the use of medicinal leeches. The authors performed an experiment designed to find a safe and practical way to sterilize the leech gut of pathogenic organisms. Leeches were incubated for a 12-hour period in solutions of antibiotic effective against A. hydrophila. The incubations in the antibiotic solutions failed to eradicate pathogenic bacteria from the gut of the leeches. The authors examined cultures of bacteria isolated from the guts of the commonly used Hirudo medicinalis (European leech) and found a wide variety of pathogenic organisms. A. hydrophila is widely believed to be the most common enteric pathogen, but the authors found A. sobria more frequently in their experiment. They also cultured the guts of the leech H. michaelseni recently used clinically in South Africa. A. caviae was the most common pathogen encountered in these leeches. A. caviae and A. sobria cause a spectra of disease similar to A. hydrophila. The authors endorse the current recommendation that all patients who have leech therapy for congested flaps or replants receive broad-spectrum prophylactic antibiotics. This appears to be the safest and simplest way to prevent leech-related infections.

The natural history of the growth of the hand: I. Hand area as a percentage of body surface area.

The use of a patients own hand as a tool to estimate the area of burn injury is well documented. The area of the palmar surface of one hand has been estimated to be 1 percent of the body surface area. The area of the palmar surface of the hand was measured to test the accuracy of this estimate and then compared with the body surface area as calculated by formulas in common use. This study also sought to determine the natural history of the growth of the hand to permit development of a readily available, bedside means of estimating hand area and body surface area. Bilateral hand tracings were obtained from 800 volunteers ranging in age from 2 to 89 years. The area of each tracing was determined using an integrating planimeter. The height and weight of each individual were measured, and his/her body surface area was calculated. The palmar hands percentage of body surface area was determined by calculating the quotient for hand area divided by body surface area. Additionally, the width of the hand was measured from the ulnar aspect at the palmar digital crease of the small finger to the point where the thumb rested against the base of the index finger. The length of the hand was measured from the middle of the interstylon to the tip of the middle finger. These two figures were multiplied together to obtain a product which approximated the area of the hand. Based on the most commonly used DuBois formula for calculating body surface area, the area of palmar surface of the hand corresponds to 0.78 ± 0.08 percent of the body surface area in adults. The percentage varies somewhat with age and reaches a maximum of 0.87 ± 0.06 percent in young children. Multiplying the length of the hand by its width overestimates the area of the hand as determined by planimetry by only 2 percent. A patients own hand may be used as a complementary, readily available template for estimation of burn area or other areas of disease or injury. In adults, the area of tracing of the outline of the hand is 0.78 percent of the body surface area, whereas in children, this number tends to be slightly higher. In the emergency room or on the wards, a simple product of length multiplied by width of the hand will closely approximate the area as determined by planimetry. This method allows a more accurate determination of the area of the palmar surface of the hand than the 1 percent estimate, which may lead to an overestimation of the size of a burn wound in adults. (Plast. Reconstr. Surg. 107: 726, 2001.)

Effects of interleukin‐8 on granulation tissue maturation

The inflammatory α‐chemokine, interleukin‐8 (IL‐8), affects the function and recruitment of various inflammatory cells, fibroblasts, and keratinocytes. Gap junctions are anatomical channels that facilitate the direct passage of small molecules between cells. The hypothesis is that IL‐8 enhances gap junctional intercellular communication (GJIC) between fibroblasts in granulation tissue, which increases the rate of granulation tissue maturation. In vitro, human dermal fibroblasts were incubated with IL‐8 prior to scrape loading, a technique that quantifies GJIC. Polyvinyl alcohol (PVA) sponges were implanted within subcutaneous pockets in rats and received local injections of either IL‐8 or saline and were harvested on day 11. In vitro, IL‐8 treated fibroblasts demonstrated an increase in GJIC by scrape loading compared to saline treated controls. In vivo, IL‐8 treated PVA sponges demonstrated a decrease in cell density and an increase in vascularization compared to saline controls by H&E staining. Polarized light viewed Sirius red‐stained specimens demonstrated greater collagen birefringence intensity, indicating thicker, more‐mature collagen fibers. IL‐8 increases GJIC in cultured fibroblasts and induces a more rapid maturation of granulation tissue.

Calmodulin‐myosin light chain kinase inhibition changes fibroblast‐populated collagen lattice contraction, cell migration, focal adhesion formation, and wound contraction

Wound healing requires fibroblast migration, synthesis of new extracellular matrix, and organization of that matrix, all of which depend upon myosin ATPase activation and subsequent cytoplasmic actin‐myosin contraction. Myosin ATPase activity is optimized by phosphorylation of myosin light chain at serine 19. Several different signaling pathways can perform that phosphorylation, the focus here is calcium saturated calmodulin dependent ‐myosin light chain kinase (CaM‐MLCK). It is proposed that CaM‐MLCK phosphorylation of myosin light chain and subsequent myosin ATPase activation affects granulation tissue fibroblast behavior and contributes to wound contraction. Myosin ATPase activity generates actin‐myosin contraction within fibroblasts. Myosin ATPase activity is involved in ATP‐induced cell contraction, the generation of focal adhesions, fibroblast migration, fibroblast populated collagen lattice (FPCL) contraction, and wound contraction. The MLCK inhibitors ML‐9 and ML‐7 inhibited ATP‐induced cell contraction, fibroblast migration, FA formation, and FPCL contraction. The calmodulin inhibitors W7 and fluphenazine blocked rat open wound contraction. In addition, fluphenazine delayed re‐epithelialization. These findings support the idea that fibroblast CaM‐MLCK activity is essential for tissue repair. We speculate that inhibition of CaM‐MLCK may reduce or prevent detrimental fibrotic contracture.

Rat mast cells communicate with fibroblasts via gap junction intercellular communications.

Usually mast cells (MCs) modulate other cellular activities through the release of their cytoplasmic granules. Recently, gap junctional intercellular communication (GJIC) between an established human MC cell line (HMC‐1) co‐cultured with human dermal fibroblasts in fibroblast populated collagen lattices (FPCLs), enhanced the rate and degree of FPCL contraction. However, HMC‐1 cells were unable to generate GJIC with human neonatal fibroblasts in monolayer culture. Here freshly isolated rat peritoneal MCs are co‐cultured with fibroblasts in collagen lattices and in monolayer culture in vitro and introduced into rat polyvinyl alcohol (PVA) sponge implants in vivo. Co‐cultured MC‐FPCL contracted faster and to a greater degree. Loading Calcein AM green fluorescent dye into red fluorescent Dil tagged MC generates MC‐paratroopers. When MC‐paratroopers form GJIC with fibroblasts, some green dye is passed into the fibroblast, while the MC‐paratrooper retains both its red and green fluorescence. MC‐paratroopers passed green fluorescent dye into both human and rat dermal fibroblasts in monolayer culture. In rats 7‐day‐old subcutaneous PVA sponge implants, which received an injection of MC‐paratroopers, exhibited auto‐fluorescent green fibroblasts, when harvested 24 h later. MC‐paratroopers pretreated with a long‐acting GJIC inhibitor prior to their introduction into PVA sponge implants, failed to pass dye into fibroblasts. It is proposed that GJIC between granulation tissue fibroblasts and MCs can modulate some aspects of wound repair and fibrosis. J. Cell. Biochem. 100: 1170–1177, 2007.

Dynamic changes appearing in collagen fibers during intrinsic tendon repair.

Intrinsic healing of severed tendons shows a delay in a gain in breaking strength and the tendon becomes translucent. The cause of tendon translucence was investigated in suture-repaired rat Achilles tendon. The repair site with adjacent translucent tendon were evaluated histologically on day 10 by immunofluorescence and transmission electron microscopy. The healing tendon translucent region by hematoxylin–eosin staining had few inflammatory cells, polarized light birefringence showed thinner collagen fibers, immunofluorescence showed few myofibroblasts, and transmission electron microscopy revealed frayed, irregular thin collagen fibers. During embryogenesis, tendon fibers grow by the addition of discreet collagen fibril segment structures. The speculation is that collagen fibril segment structures are released from collagen fibers within the translucent tendon region for reuse during the regeneration of tendon collagen fibers during intrinsic tendon repair. Healing tendon translucence is related to a decrease in the diameter of collagen fibers by the release of collagen fibril segments within tendon bundles/fascicles.

Incidence of operative procedures on cleft lip and palate patients.

The incidence of operative procedures in a group of 374 cleft lip and palate patients was determined by a chart review. These patients were part of a long term study funded by the National Institute of Dental Research at the Lancaster Cleft Palate Clinic. The chart review provided a breakdown of the primary and secondary procedures performed on the patients. Although the secondary procedures carried out were underestimated in this study, it is clear that these operations comprise a major component of the successful treatment of these patients. The results of the study and the implications in todays managed care environment are discussed.

Gap junction communications influence upon fibroblast synthesis of Type I collagen and fibronectin

In rats polyvinyl alcohol sponge subcutaneous implants treated with gap junctional intercellular communications (GJIC) uncouplers showed reduced deposition of connective tissue. Do uncouplers inhibit the synthesis and deposition of a new connective tissue by fibroblasts? Confluent human dermal fibroblasts in serum‐free medium received either endosulfan or oleamide, GJIC uncouplers. Collected media were subjected to Dot Blot analysis for native Type I collagen and fibronectin. Uncoupler‐treated fibroblasts released less Type I collagen, while there was no change in fibronectin release. Collagen synthesis was restored to normal, when the uncouplers were removed, showing that these uncouplers were reversible and not toxic to cells. Northern blot analysis revealed procollagen α1 (I) mRNA was minimally affected by endosulfan. Oleamide‐treated 17‐day chick embryo calvaria explants were incubated with Type I collagen antibody, frozen, cryosectioned, and then subjected to rhodamine (Rh) tagged anti‐mouse‐IgG antibody, to detect newly deposited Type I collagen. Fluorescent antibody‐collagen complexes were localized on the periphery of cells in control calvaria, but absent around cells in oleamide‐treated calvaria. GJIC optimize collagen synthesis but not fibronectin synthesis. The lack of connective tissue deposited in granulation tissues treated with uncouplers appears related to the inhibition of collagen synthesis. These findings suggest that altering GJIC might control collagen deposition in scarring. J. Cell. Biochem. 98: 735–743, 2006.