Mitogenic and Protein Synthetic Activity of Tissue Repair Cells: Control by the Postsurgical Macrophage

It is well known that fibroblasts are a main source of extracellular matrix synthesis necessary for tissue repair. In addition, macrophage s secrete products that are known to modulate synthesis of extracellular matrix. Accordingly, we studied the incorporation of [³H]thymidine, [³H]proline, and [³⁵S]sulfate into macromolecules produced by fibroblasts recovered from the site of peritoneal tissue repair cultured with and without spent media from postsurgical peritoneal macrophages. Rabbits underwent resection and re-anastomosis of their small intestines. Peritoneal exudative cells (PEC) were then collected on postsurgical day 5 and day 10 as well as from nonsurgical controls, separated by discontinuous Percoll gradient centrifugation, and cultured for 48 h. A second group of rabbits underwent peritoneal wall abrasion from which fibroblast tissue repair cells (TRC) were collected from the site of injury at postsurgical day 7 and maintained in culture for varying times. Incorporation of radiolabeled precursors into DNA, collagen, and sulfated proteoglycans was determined. Incorporation of [³H]thymidine and [³H]proline into untreated TRC gradually decreased with culture duration. Conversely, [³⁵S]sulfate incorporation gradually increased during prolonged culture. Macrophage spent media increased the levels of [³H]thymidine incorporation by the TRC. [³H]Proline and [³⁵S]sulfate incorporation into TRC were also stimulated by macrophage spent media. However, this stimulation may be due to the enhanced proliferation of TRC by macrophage spent media. In conclusion, tissue repair fibroblasts are activated for postsurgical repair at the site of injury by many factors including secretory products from postsurgical macrophages.     

Modulation of proline and glucosamine incorporation into tissue repair cells by peritoneal macrophages

The purpose of this study was to determine the patterns of [14C]proline and [14C]glucosamine incorporation by tissue repair cells (TRC) as modulated by postsurgical macrophages. Rabbits underwent a midline laparotomy followed by resection (2.0 cm) and reanastomosis of their ileum. Another group of rabbits underwent peritoneal wall abrasion with sterile gauze until punctate bleeding developed. Postoperative (1-28 days) exudate cells (PEC) were recovered from the peritoneal cavity after reanastomosis, and (TRC) were obtained directly from the injured peritoneal surface after abrasion. Since the postsurgical exudate was composed mainly of macrophages, we examined the effect of postsurgical macrophage-spent media on the incorporation of [14C]proline, [14C]glucosamine, and [3H]thymidine by TRC. After 7 days of culture, Postsurgical Day 7 TRC were incubated with spent media from postsurgical PEC (greater than 90% macrophages). When TRC were cultured with macrophage-spent media, the number of TRC increased significantly compared to that of fresh medium-treated controls. The incorporation of [3H]thymidine by TRC was also enhanced by macrophage-spent media. The incorporation of [14C]proline and [14C]glucosamine by TRC was also enhanced when incubated with macrophage-spent medium. However, when data were expressed on a per cell basis, incorporation of [14C]proline and [14C]glucosamine by TRC cultured with macrophage-spent media was the same or less than that by cells incubated with fresh medium. These data suggest that the increase in incorporation of glucosamine and proline into connective tissue protein by postsurgical repair cells may be directly modulated by macrophages recruited in response to surgical injury and that this increase is due to the fibroproliferative effect of postsurgical macrophages.     

Regulation of proliferation of peritoneal tissue repair cells by peritoneal macrophages

Macrophages produce soluble mediators which modulate fibroblast growth during tissue repair. Interaction between tissue repair fibroblasts (TRC) and regulatory proteins from surgically elicited macrophages is important for peritoneal reepithelialization. In this study, we compared the effects of an extract from postsurgical macrophage spent medium with those of known growth factors on TRC collected from injured peritoneum to evaluate certain characteristics of macrophage secretory products on peritoneal healing. Rabbits underwent a midline laparotomy followed by resection and reanastomosis of the ileum or abrasion of the abdominal wall. TRC were then collected at various times after surgery. Peritoneal macrophages recovered from nonsurgical or postsurgical rabbits were cultured for 2 days in vitro. The peak of thymidine incorporation by TRC occurred on day 5 after surgery; this gradually decreased with extended postsurgical times. Fibroblast growth factor and epidermal growth factor stimulated, whereas TGF-beta inhibited, [3H]thymidine incorporation into TRC. Maximal thymidine incorporation occurred when TRC from Postsurgical Day 5 were cultured with an extract from postsurgical macrophage spent medium. However, when TRC recovered from Postsurgical Days 2 and 10 were cultured with an extract of postsurgical macrophage spent medium, they showed greater stimulation than Day 5 TRC. These data suggest that postsurgical macrophages may produce an array of factors that stimulate fibroblast growth and differentiation and may in turn affect tissue repair throughout the wound healing process.     

Incorporation of thymidine by fibroblasts: evidence for complex regulation by postsurgical macrophages

Macrophages and fibroblasts are major components of the postsurgical repair process. In order to understand more fully the interaction between these two cell types, we studied the modulation by macrophages of the incorporation of [3H]thymidine into postsurgical fibroblasts recovered from the site of peritoneal injury. Peritoneal exudate cells (PEC):(greater than 95% macrophages) were collected from rabbits 4 and 7 days after resection and reanastomosis of the small intestine. PEC were suspended in Medium 199 (M-199) with 3% fetal calf serum (FCS) and incubated for 48 hr. Fibroblasts were obtained from rabbits that underwent abrasion of the parietal peritoneum 7 days previously, and were cultured for 7 days in M-199 with 3% FCS. Fibroblasts were then replated and incubated with macrophage-spent medium. Incorporation of [3H]thymidine into fibroblasts was significantly suppressed after 24 hr of incubation with macrophage-spent media compared to the incorporation by fibroblasts incubated with fresh medium (control). This suppression was most profound when fibroblasts were incubated with resident (nonsurgical) macrophage-spent medium. The incorporation of thymidine by macrophage-spent media groups then increased rapidly and reached control levels at 48 hr of incubation. After 54 hr of incubation, the incorporation of thymidine by fibroblasts incubated with media from postsurgical macrophages was significantly higher than that of control. Morphological changes in fibroblasts also appeared as the culture with macrophage-spent media progressed. Initially, fibroblasts were shaped like pine needles, but after 7 days of culture, fibroblasts assumed a spherical shape. Round-shaped fibroblasts returned to the original morphology (pine needle shape) after incubation for 48 hr with macrophage-spent medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of fibroblast proliferation by postsurgical macrophages

Macrophages and fibroblasts are major components of postsurgical peritoneal repair. In order to understand the interaction between these two cell types, we studied the effects of spent macrophage culture media on fibroblast proliferation. Rabbits underwent resection and reanastomosis of their small intestine. Peritoneal exudative cells (PEC) were then collected from these animals on postoperative Days 4, 7, and 28 and from nonsurgical controls. PEC (5 X 10(5) cells/ml) were cultured in M-199 with 3% fetal calf serum. After 48 hr the spent media from the cultured PEC were harvested, centrifuged (200g for 10 min), and stored (medium: M-D0, D4, D7, D28). A second group of rabbits underwent peritoneal wall abrasion followed by collection of fibroblasts directly from the site of injury on postoperative Days 1, 4, and 7. After Day 7 of culture, fibroblasts were resuspended and seeded into dishes (1 X 10(5) cells in 1 ml medium), to which was added 1 ml of spent PEC culture medium. After 24 hr of incubation, 1 muCi [3H]thymidine was added for an additional 18 hr. Fibroblasts were then collected and the amount of [3H]thymidine incorporated into trichloroacetic acid-precipitable material was quantitated. In one protocol, fresh M-199 with 3% fetal calf serum was used, and in another protocol, "U-medium" (which was previously incubated for 48 hr with fibroblasts) was used. The cytology of the PEC was determined by Wright's staining, nonspecific esterase activity, and phagocytosis. At least 80% of the peritoneal exudative cells were identified as macrophages. Postsurgical Day 7 fibroblasts demonstrated greater [3H]thymidine incorporation compared to fibroblasts from postoperative Days 1 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mitogenic activity of peritoneal tissue repair cells: control by growth factors

The purpose of this study was to determine the proliferative activity of tissue repair fibroblasts recovered directly from injured peritoneum at various times after surgery and to test the mitogenic response of tissue repair cells (TRC) to growth factors. Rabbits underwent bilateral peritoneal abrasion (5 X 5 cm) with sterile gauze until punctate bleeding developed. Postsurgical (Days 2, 5, 7, and 10) tissue repair cells were recovered from the injured peritoneum by scraping with a scalpel blade. Although tissue repair cells consisted of a mixed cell type after 4 days in culture, recovered cells were essentially fibroblasts. These TRC were then pulsed with [3H]thymidine after 4 days in culture. The incorporation of thymidine into Postsurgical Day 5 TRC increased significantly compared to that of Day 2 TRC (P less than 0.05). Incorporation then decreased with time following surgery. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) stimulated the incorporation of thymidine into TRC. However, the response of Postsurgical Day 7 and 10 TRCs to 1 microgram/ml EGF was significantly greater than those of Postsurgical Day 2 and 5 TRCs (Day 2 TRC, 166 +/- 7.4; Day 10 TRC, 420 +/- 96% of control cells without EGF, P less than 0.05). Platelet-derived growth factor (PDGF, 10 ng/ml) also stimulated the incorporation of thymidine into Day 10 TRCs, but this stimulatory activity (129.9 +/- 8.5% of control) was less than EGF or FGF. IL-1 alpha and IL-2 did not stimulate the incorporation of thymidine into TRC at a concentration of 100 pg/ml, but these cytokines did stimulate protein synthesis by TRC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of peritoneal re-epithelialization by postsurgical macrophages.

The studies reviewed here show that postsurgical macrophages are capable of modulating the proliferation of TRC. That is, macrophages either suppress or enhance the proliferation of TRC depending on the culture time and the medium used as a comparison, i.e., culture medium with only serum or spent medium from cultures of resident peritoneal macrophages. Postsurgical macrophages also modulate the morphology of (spindly or rounded appearance) and the secretion of extracellular matrices by TRC. The responsivity of TRC to control by postsurgical macrophage-spent media or growth factors changes as a function of postsurgical and/or culture time. In addition, cells harvested from the site of peritoneal trauma (TRC) did not respond to growth factors in a fashion entirely the same as fibroblasts. This indicates that cells harvested from the site of peritoneal injury are unique. Lastly, after removal of a suppressive factor from postsurgical macrophage-spent media by dialysis, the factors secreted by postsurgical macrophages are more potent in enhancing TRC proliferation than growth factors individually.     

Proliferative and synthetic response of bovine growth plate chondrocytes to various capacitively coupled electrical fields

In vitro monolayer cultures of growth plate chondrocytes isolated from newborn calf costochondral junctions were subjected to capacitively coupled electrical fields for 48 h. In part A, the electrical signal was a 60-kHz sine wave applied at different voltages so as to produce electrical fields at the pericellular level of 7, 20, 50, and 126 mV/cm. Incorporations of [3H]thymidine and [35S]sulfate were assayed to determine the effect of the above fields on cells proliferation and matrix synthesis, respectively. Proliferation was increased by 47% in the 20 mV/cm field whereas the same field decreased [35S]sulfate incorporation by 21%. These changes were significant at p less than 0.05 in both instances. In part B, the 20 mV/cm field was applied in a pulsed fashion to produce daily duty cycles of 100, 25, 2, and 0.25%. Incorporation of [3H]thymidine, [35S]sulfate, and [14C]proline per DNA were assayed. Results indicated that the 100, 25, and 0.25% percent duty cycles showed significantly (p less than 0.01-0.05) increased proliferation, whereas the 0.25% signal (5 ms on/495 ms off for 6 h/day) significantly decreased [14C]proline incorporation. We conclude that the biologic response of cells in vitro is signal specific, and that the total amount of electrical energy required to stimulate the growth plate chondrocyte to increased proliferation is very small since the total time the 0.25% duty cycle signal was only 3.6 min of a 24-h period.     

Integrative cartilage repair: adhesive strength is correlated with collagen deposition.

Procedures to repair focal articular cartilage defects often result in poor integration between the host cartilage and the graft tissue, and this may be related to the lack of matrix deposition and the death of chondrocytes near a cut cartilage surface. The objective of this study was to determine if cartilage repair was related to deposition of newly synthesized collagen. The mechanical integration that occurred between two live adult bovine cartilage blocks cultured in partial apposition for two weeks was correlated with [3H]proline incorporation, a measure of protein synthesis, of which more than 66% was accounted for by collagen. A similar level of mechanical integration occurred in sample pairs consisting of a live and killed cartilage block, and this adhesive strength was also correlated with [3H]proline deposition into both the live and the killed blocks. In these samples, the [3H]proline deposited into the killed cartilage appeared to originate from chondrocytes in the live cartilage, since live cells were not detected in the killed cartilage block by either viability staining or [35S]sulfate incorporation. These results suggest a mechanism of integrative cartilage repair in which live chondrocytes within cartilage secrete matrix molecules that are components of a collagen network, and subsequent deposition of these molecules near the repair interface contributes to functional integration.     

In vitro growth of bovine articular cartilage chondrocytes in various capacitively coupled electrical fields

Isolated articular cartilage chondrocytes from 1- to 3-week-old male Holstein calf knee joints were formed into pellets containing 4 X 10(6) isolated cells and were grown in tissue culture medium (minimum essential medium/NCTC 135) containing either 1 or 10% newborn calf serum (NBCS) in plastic Petri dishes in 5% CO2 and air at 37 degrees C in saturation humidity. On the 4th postisolation day either [35S]sulfate or [3H]thymidine was added to the medium, and the pellets were exposed for 24 h to capacitively coupled electrical fields (10, 100, 250, and 1,000 V peak-to-peak, 60 kHz, sine wave signals). The pellets were then harvested, dialyzed, hydrolyzed, and assayed for DNA, protein, [35S]sulfate incorporation, and [3H]thymidine incorporation. Results indicated that at 250 V peak-to-peak there was a statistically significant increase in [35S]sulfate in 1% NBCS and a statistically significant increase in [3H]thymidine in 10% NBCS. At potentials above or below 250 V no changes were noted. Thus, articular cartilage chondrocytes grown in pellet form can be stimulated to increase glycosaminoglycan synthesis or to increase cell proliferation by an appropriate capacitively coupled electrical field. The importance of the serum concentration in the medium in evaluation of biosynthesis in vitro is noted.     

A role of postsurgical macrophage activation by peritoneal injury]

At a site of peritoneal injury after abdominal surgery, macrophages are thought to be a principle type of inflammatory cells. Therefore, we determined the metabolic activities of postsurgical peritoneal exudate macrophages using standardized rabbit model. Rabbits underwent midline laparotomy followed by resection and reanastomosis of the ileum. At various days after surgery, peritoneal exudate macrophages were recovered from lavage fluid. Postsurgical Day-5 macrophages expressed significantly high potential to produce superoxide anion even without PMA stimulation compared to non-surgical control macrophages, although an activity of Day-10 macrophages was similar to control. The conditioned media from postsurgical Day-1 macrophage culture strongly inhibited urokinase type plasminogen activator (PA) and this plasminogen activator inhibitor (PAI) activities decreased following the extension of postsurgical time. Conversely, PA activities of macrophage-conditioned media decreased by day 1 and then gradually increased reaching control levels by day 10. Elastase activities of macrophage-conditioned media gradually decreased until postsurgical day 10. These data suggest that surgical injury activates postsurgical exudate macrophages. However, a time course of metabolic activities of these cells was dependent upon each secretory products. This differential secretion might express the stage of activation and differentiation of postsurgical macrophages. Moreover, postsurgical activated macrophages may control the tissue repair through a digestion of injured matrix and fibrinolytic process.     

Modulation of postsurgical macrophage function by postsurgical polymorphonuclear leukocytes]

Rapid and transient influx of polymorphonuclear leukocytes (PMN) is observed prior to accumulation of macrophages after surgical trauma. Rabbits underwent intestinal reanastomosis and at various times peritoneal exudate cells were collected and separated using a Percoll gradient. Postsurgical macrophages were incubated with PMN spent media obtained from various postsurgical periods. Macrophage release of O2- had already increased at 2 hr after surgery, reached peak levels at 6 hr and decreased by 24 hr. PMN spent media from 6 hr postsurgical cells functioned as a suppressor, whereas 12 or 24 hr PMN spent media increased the O2- release from the macrophages harvested at 6 and 12 hr after surgery. Plasminogen activator (PA) activity in the macrophage spent medium was elevated at 24 hr after surgery by exposure to PMN spent media, however no effects were observed on macrophages harvested within 12 hr after surgery. PA inhibitory activity was reduced at 2 hr after surgery, and gradually increased, but no effects of PMN spent media on the PA inhibitory activity was observed. Thus, soluble factors secreted into the medium by PMN may modulate macrophage metabolism in stages as the macrophages differentiate and promote wound repair.     

Possible genetic defects in regulation of glycosaminoglycans in patients with diabetic nephropathy

The hypothesis of genetic defects in glycosaminoglycan (GAG) regulation among patients with insulin-dependent diabetes mellitus (IDDM) and nephropathy was assessed by studies in tissue cultures of fibroblasts obtained from 7 patients with normal urinary albumin excretion, 11 patients with diabetic nephropathy, and 6 nondiabetic control subjects. The incorporation of [2H] glucosamine and [35S] sulfate into hyaluronic acid (HA), chondroitin sulfate and dermatan sulfate (CS + DS), and heparan sulfate (HS) was measured in cells, matrix, and medium and related to micrograms of tissue protein. Large interindividual variations were seen in all three groups, and the incorporation of [3H] glucosamine into HA, CS + DS, and HS and [35S] sulfate into CS + DS and HS were not significantly different between the three groups. However, the fractional incorporation of [3H]glucosamine into HS was significantly reduced in diabetic patients with nephropathy compared with control subjects. This was the case not only when related to the total amount of GAGs (P = 0.014) but also when related to HA (P = 0.014). No significant difference was seen between control subjects and normoalbuminuric diabetic patients. The degree of N-sulfation of HS was not significantly different between the experimental groups. The results suggest that patients with diabetic nephropathy may suffer from deficiencies of coordinate regulation in the biosynthesis of GAG in fibroblasts, which may lead to a reduced density of HS in the extracellular matrix. If these changes reflect alterations in the biosynthesis of GAG from endothelial, myomedial, and mesangial cells, this observation may be relevant for the pathogenesis of severe diabetic complications.     

Kinetic analysis of experimental post-operative peritoneal healing: the incorporation of proline and glucosamine by exudative and tissue repair cells.

The purpose of this study was to experimentally investigate the cellular composition of post-surgical peritoneal fluid and peritoneal tissue and determine the patterns of 14C-proline and 14C-glucosamine incorporation by the peritoneal exudative cells and peritoneal tissue repair cells (PEC and PTRC). One group of rabbits underwent resection (2.0 cm) and reanastomosis of their ileum, and another group underwent peritoneal wall abrasion. Postoperatively (1-28 days), the PEC and PTRC were collected and incubated for 5 days with 0.5 mu Ci 14C-glucosamine or 14C-proline, and the specific activity thereafter determined by beta counting. On the 1st postoperative day, the total cell number (TCN) has increased (7.7 x 10(7) cells/rabbit) to 770 per cent of the control values primarily as a result of the PMN influx (89.9 per cent). On day 3, the TCN was 6.1 x 10(7), 58.5 per cent of which comprised macrophages, which had become the principle cell type by day 5. The incorporation of proline and glucosamine into the PEC increased significantly peaking on day 7 then decreasing to the control value by day 21. Proline incorporation into the PTRC increased significantly, reaching a peak value on day 5, which decreased by day 10. Glucosamine incorporation reached a peak value on day 7 then decreased by day 10. In conclusion, the increase in glucosamine and proline incorporation into the PTRC parallels the increase in PEC, comprised principally of macrophages. These findings suggest that analysis of the metabolic activities in peritoneal activated macrophages may provide a useful tool to dissect the central mediation of postsurgical peritoneal re-epithelialization.     

Mechanical and physiochemical determinants of the chondrocyte biosynthetic response

The relation between mechanical loading of cartilage and chondrocyte activity in vivo may be mediated by several physical transduction mechanisms including: cell deformation, hydrostatic pressure gradients, fluid flow, streaming currents, and physicochemical changes. We have developed an organ culture system designed to study chondrocyte biosynthetic response to such physical stimuli. This study focuses on the effects of static compression and physicochemical changes. Cartilage disks harvested from the reserve zone of the epiphyseal plate of 1-2-week-old calves were subjected to static compressive stresses of 0-3 MPa in unconfined compression and the incorporation of [35S]sulfate and [3H]proline was measured during the 12-h loading period. Incorporation of both proline and sulfate decreased monotonically with increasing stress. Compressive loading also produces physicochemical changes including a decreased water content and increased matrix fixed-charge density, with a concomitant increase in interstitial counterion concentrations (e.g., K+, H+) and decreased coion concentrations (e.g., SO4(2-). We therefore examined the possibility that specific changes in interstitial mobile ion concentrations may be linked to chondrocyte response to static compression by measuring biosynthesis in the absence of mechanical compression while independently altering the SO4(2-), K+, and H+ composition of the bathing medium. We found that proline and sulfate incorporation were strongly dependent on pH, but independent of [SO4(2-)] and [K+] in the range studied. These results suggest that compression-induced changes in local, interstitial pH may account for the observed biosynthetic response to static compression.     

Kinetic analysis of experimental post-operative peritoneal healing: The incorporation of proline and glucosamine by exudative and tissue repair cells

The purpose of this study was to experimentally investigate the cellular composition of post-surgical peritoneal fluid and peritoneal tissue and determine the patterns of¹⁴C-proline and¹⁴C-glucosamine incorporation by the peritoneal exudative cells and peritoneal tissue repair cells (PEC and PTRC). One group of rabbits underwent resection (2.0 cm) and reanastomosis of their ileum, and another group underwent peritoneal wall abrasion. Postoperatively (1–28 days), the PEC and PTRC were collected and incubated for 5 days with 0.5 μCi¹⁴C-glucosamine or¹⁴C-proline, and the specific activity thereafter determined by beta counting. On the 1st postoperative day, the total cell number (TCN) had increased (7.7×10⁷ cells/rabbit) to 770 per cent of the control values primarily as a result of the PMN influx (89.9 per cent). On day 3, the TCN was 6.1×10⁷, 58.5 per cent of which comprised macrophages, which had become the principle cell type by day 5. The incorporation of proline and glucosamine into the PEC increased significantly peaking on day 7 then decreasing to the control value by day 21. Proline incorporation into the PTRC increased significantly, reaching a peak value on day 5, which decreased by day 10. Glucosamine incorporation reached a peak value on day 7 then decreased by day 10. In conclusion, the increase in glucosamine and proline incorporation into the PTRC paralles the increase in PEC, comprised principally of macrophages. These findings suggest that analysis of the metabolic activities in peritoneal activated macrophages may provide a useful tool to dissect the central mediation of postsurgical peritoneal re-epithelialization.     

Collagen synthesis in cultured mesothelial cells. Response to silica

Mesothelial cells were isolated from the peritoneal surface of rats using trypsinization. The cells were polygonal-shaped and proliferated rapidly forming confluent cultures. Application of colloidal silica to mesothelial cell cultures in doses known to induce peritoneal adhesion disease was injurious to the cells and reduced their synthesis of total proteins and collagen. This effect was dose dependent. Media from silica-treated mesothelial cells and granulation tissue fibroblasts were applied to similar but non-treated mesothelial cell and granulation tissue fibroblast cultures. Media from SiO2-treated mesothelial cells increased collagen synthesis markedly both in the mesothelial cell and fibroblast cultures compared with the control cultures as estimated from the incorporation of radioactive proline into hydroxy-proline-containing proteins. However, no stimulation of collagen synthesis was induced by media from the SiO2-treated granulation tissue fibroblasts. Similar effects are known to be induced in fibroblast cultured by media from silica-treated peritoneal macrophage cultures. The alkaline ribonuclease activities of the media were decreased in silica-treated mesothelial cell and macrophage cultures but not in the media of fibroblast cultures. These results suggest that mesothelial cells, like macrophages, can interfere with the protein synthesis of fibroblasts and thus contribute to the regeneration of mesothelium after injury and to the formation of adhesions.     

Studies of cultured human fibroblasts in diabetes mellitus: changes in heparan sulfate.

The incorporation of [35S]sulfate into glycosaminoglycans was studied in cultures of normal and diabetic skin fibroblasts. Heparan sulfate was determined by column chromatography after enzymatic degradation of chondroitin sulfates and dermatan sulfate by chondroitinase ABE. Cultured skin fibroblasts from both insulin-dependent and noninsulin-dependent diabetics were found to have increased proportions of heparan sulfate in the media relative to the other sulfated glycosaminoglycans.     

Effects of calcitonin on wound healing: A morphological study in rabbits

The cellular and metabolic effects induced by calcitonin on regenerative processes during wound healing were studied in rabbits. Electron microscopy revealed marked changes at 2 and 5 days postwounding in epidermal cells and fibroblasts in calcitonin-treated rabbits such as: an increased in keratohyaline granules, tonofilaments, and polysomes and hypertrophy of nuclei in the epidermal cells. Fibroblasts are hypertrophied and surrounded by large areas of mature collagen fibers at 5 and 14 days. Electron microscopic autoradiography revealed an increase in the incorporation of [³H]thymidine mostly in the nuclear chromatin, [³H]uridine in the nucleoli, [³H]leucine in the endoplasmic reticulum, polysomes, and newly formed keratin layers, and [³H]proline over fibroblasts and collagen fibers. Scanning electron microscopy showed changes in the collagen fibers and keratin pattern. The collagen fibers are hypertrophied, with an extensive network of fibrils in calcitonin-treated rabbits. A typical scaly pattern of keratin can be noted in calcitonin-treated rabbits while in controls it is smooth and homogenous. These findings demonstrate that calcitonin stimulates protein synthesis, keratinogenesis and collagen formation in epidermal cells and fibroblasts and thus accelerates wound repair.     

Kinetics of interleukin-1 secretion by murine macrophages recovered from the peritoneal cavity after surgery

Macrophages play a central role in wound healing after surgical injury possibly through the secretion of soluble factors like interleukin-1 (IL-1). IL-1 has many biological activities which regulate most of the cell types that appear in postsurgical wounds. In this study, we determined the levels of IL-1 production by murine macrophages harvested from postsurgical exudate at several time points after standardized peritoneal surgery. To further characterize the level of functional activities of postsurgical macrophages, the IL-1 levels were determined with or without stimulating the cells with lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). After surgery, the number of macrophages harvested by peritoneal lavage increased, reached peak levels on Postsurgical Day 3, and then decreased. IL-1 levels secreted by macrophages cultured without stimuli gradually increased after surgery, reaching peak levels on Day 10. In conditioned media from LPS-PMA-stimulated macrophages, IL-1 levels were significantly greater than in media from unstimulated macrophages and peaked on Postsurgical Day 3. These data suggest that the susceptibility of postsurgical macrophages to stimuli changes during the postsurgical wound healing process. Accordingly, IL-1 may play an important role in the late rather than in the early phase of peritoneal repair.