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Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat

^a Department of Pathology, Vanderbilt University and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212, USA; and

<sup>b

c</sup> Center for Cell Research, The Pennsylvania State University, University Park, Pennsylvania 16802-6005, USA

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at 22°C and 28°C. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. Growth factor responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both factors in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of factor release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.—Davidson, J. M., Aquino, A. M., Woodward, S. C., Wilfinger, W. W. Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat.

Key Words: space medicine • granulation tissue • PDGF • bFGF

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
LITTLE IS KNOWN ABOUT wound healing in a microgravity environment, despite the likelihood that increasing use of manned spaceflight as a research and commercial enterprise raises the probability of traumatic injury in this state. Published studies of wound repair in space have been confined to repair of bone or muscle, two tissues that depend on biomechanical stimulation for proper repair (1 , 2 ). Before this experiment, there were no published studies of cutaneous wound repair during spaceflight. Wound healing consists of a complex interaction of soluble factors, extracellular matrix, inflammatory and tissue cells. The normal repair sequence includes inflammation and cellular migration, followed by cellular proliferation and connective tissue synthesis (3) . Impaired healing can arise from a myriad of circumstances, including systemic conditions such as immunosuppression by glucocorticoids, chemotherapeutics, vitamin or mineral deficiencies, and the diabetic state (4) . When sponge discs such as polyvinyl acetal are subcutaneously implanted beneath the ventral panniculus carnosus, adjacent to the abdominal musculature in rats, they create a spatially defined, porous matrix into which plasma exudes, forming a fibrin clot (5) . The matrix is then progressively infiltrated by granulation tissue, the organ of repair, where collagen is elaborated. The rate of sponge organization reflects wound healing states (6) .

This study examined the intrinsic capacity of rats to form granulation tissue during spaceflight. We also determined whether model wounds would respond normally to stimulation by recombinant cellular growth factors. We compared the effects of slow-release pellets releasing basic fibroblast growth factor (bFGF)³ and platelet-derived growth factor (PDGF-BB), both known to accelerate granulation tissue formation in the wound site (7) . Previous studies had shown that single and multiple injections of these growth factors directly into wound sites such as sponges and incisional wounds were effective in accelerating granulation tissue formation (8 , 9 ). To achieve drug delivery while test animals were in a sealed environment, the experiment consisted of polyvinyl acetal sponge implants containing pellets designed to release these factors continuously for 14 days (10 , 11 ).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Pathogen-free adult male Fisher 344 rats weighing a mean of 206.8 g, approximately 7 wk of age, were purchased from Taconic Laboratories (Germantown, N.J.). Control and experimental animals were weight matched within each treatment group.

After intraperitoneal ketamine/xylazine anesthesia, sterile preparation, and midline abdominal incision, each animal was implanted subcutaneously with six polyvinyl acetal CF-50 sponges (a gift from Merocel, Mystic, Conn.) placed beneath the panniculus carnosus and adjacent to the abdominal musculature, as detailed elsewhere (5) . The center of each sponge contained a sustained release pellet (10) (Innovative Research of America, Sarasota, Fla.), designed to release one of the following substances at the controlled rate of 500–750 ng/day, either beginning immediately after implantation or after a 2-day delay, over a period of 14 days: 1) placebo, an unaltered pellet composed of cholesterol, cellulose, phosphates, stearates, and lactose; 2) recombinant PDGF-BB (Upstate Biotechnology, Lake Placid, N.Y.); and 3) recombinant human bFGF (Upstate Biotechnology). Implantation was done in a randomized block design to determine any positional effects on wound organization in each of the weight-matched animal pairs.

A total of 48 rats were used in this experiment (12) . An initial group of 12 animals were implanted with 6 PVA sponges/pellets and, immediately after surgical recovery, were housed in two animal enclosure modules (six rats/AEM), transported to the Space Shuttle Endeavour (STS-57; PSE-03), and placed in the Orbiter ~16.5 h prior to the anticipated launch. Because of a launch delay, the microgravity phase began 42 h after sponge implantation. Mission length was 9 days, 23 h, and 44 min. Endeavour returned to Kennedy Space Center on July 1, 1993, at 8:42 AM. The rats were removed from the hardware at ~12:30 PM. On the morning of launch (48 h after the flight animals were loaded on to the middeck of the Orbiter) another two sets of animals, 12 from the 22°C selection group and 24 from the 28°C selection group, were implanted with CF-50 sponges and growth factor pellets as previously described. Twelve animals from the 28°C group were loaded into two AEM (six rats/AEM) and transferred to the Orbital Environmental Simulator at the Kennedy Space Center Life Science Support Facility (13) . Middeck temperature, humidity, and pCO₂ data obtained from the Orbiter were used on a 48 h delay basis to emulate the AEM environment aboard the Shuttle in the AEM on the ground so that the two principal variables of the orbital flight group were reduced gravity and cosmic radiation exposure. The remaining 24 rats from the 22° and 28°C temperature groups were housed in standard vivarium cage (two rats/cage) and returned to the vivarium after the implantation procedures were completed. After a 10-day spaceflight exposure or simulation and 1 h prior to death, animals received an injection of hypothalamic-releasing hormones as part of a separate, acute study. The animals were killed, then sponges were removed from each animal and partitioned for biochemical and histological analysis. Sponges were cut into four segments, and the wet weights of the whole sponge and each segment were determined. Segments were then frozen in LN₂ or fixed in 4% paraformaldehyde for histological studies. Paraformaldehyde-fixed sponges were embedded in paraffin, sectioned, stained with hematoxylin-eosin and Masson trichrome, and examined in a blind, coded fashion. Sponges were examined for degree of organization, resolution of hemorrhage, and intensity of collagen staining. One segment was analyzed for DNA content by fluorometric assay (14) , one for protein content by the method of Bradford (15) , and one for collagen content as calculated from the concentration of hydroxyproline, measured as its phenylisothiocyanate derivative by reverse-phase high performance liquid chromatography (16) . Percentage collagen content was calculated from the relative proline and hydroxyproline values.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Methodology for morphological analysis of the sponges has been detailed elsewhere (5 , 17 ). In these studies a portion of the sponge and its pellet was sectioned vertical to its disc surface. The degree of histological organization (the infiltration of the sponges' interstices by granulation tissue, replacing fibrin and inflammatory cells), the amount of visible collagen revealed by trichrome staining, and the resolution of hemorrhage at the infiltrating interface were examined. These variables were scored, and the scores for each group were compared, using the Student-Newman Keuls statistic (18) , analyzing the effects of microgravity as well as the vulnerary effects of growth factors. Histologically, differences in collagen content values among groups were not conspicuous (data not shown). The lack of demonstrable effects on microscopically visible collagen when significant differences in collagen content values were found by chemical analysis for hydroxyproline (vide infra) confirmed the necessity for biochemical analysis (5 , 16 ).

Histological analysis was performed to ascertain the degree of organization of granulation tissue and the extent to which each variable contributed to changes in that organization (Table 1 ). Both bFGF and PDGF showed positive effects in the ground control rats, whereas only immediate-release bFGF and delayed-release PDGF produced significant, positive effects in the flight rats as compared with flight controls. The initial experimental design was based on the hypothesis that delay of growth factor release until microgravity was obtained would compensate for the interval between surgery and launch. However, this experimental design was confounded by launch postponement. Differences may have been minimized by the unforeseen 2-day delay before earth orbit began, although wound organization is minimal at this early phase. Thus, organizational responses of granulation tissue that were surprisingly normal occurred in microgravity, and the granulation tissue mounted, at least at the histological level, a positive response to wound repair-stimulating growth factors in flight animals, as occurs in normal gravity (8 , 9 ). Although the histological interpretation indicated a trend toward reduced tissue organization in microgravity, the analyses were not robust enough to establish statistical significance.

Biochemical findings were more sensitive in this model. Biochemical analysis of sponge granulation tissue established that there was no significant effect of the release rate (immediate or delayed), the implantation site, or the type of control environment, as assessed by multifactorial analysis of variance (ANOVA; data not shown). Sponge cellularity was increased significantly by both growth factors in ground controls (Fig. 1 ), and cellular influx into the tissue space of placebo-treated sponges was unaffected by spaceflight. In contrast, there was a significantly blunted response to either bFGF or PDGF-BB in flight animals (P<0.005, ANOVA; Fig. 1 ). When the analysis compared the pooled response of all flight vs. all ground animals, there was also a significant reduction or lack of response in flight animals.

View larger version (47K): [in this window] [in a new window]   Figure 1. Blunted response of granulation tissue to growth factor stimulation in microgravity. Polyvinyl acetal sponges were implanted subcutaneously in rats. Each sponge contained a slow release pellet that released the indicated material at a rate of ~1 µg/day for the duration of the experiment (12 days). The data represent the combined results from pellets with immediate and delayed (2 days) release, since ANOVA established there was no significant difference between these delivery schemes (data not shown). The flight group (shaded bars) represents data from animals (n=12) whose experimental wounds became organized during the 10-day orbit in the Space Shuttle Endeavour. Ground data represent findings from an environmentally equivalent group of animals (n=12) that were housed in an equivalent module (AEM), matched for temperature, gases, and humidity with the flight AEM, at the launch site. Portions of each implanted sponge (6 per rat) were recovered at the end of the flight from expired animals, homogenized, and analyzed for DNA content by a fluorimetric assay. Both basic FGF and PDGF-BB produced significant increases in cellularity of sponge granulation tissue (P<0.05) in the ground group of animals. The growth factors did not produce significant increases over placebo in cellularity of sponges in the flight group. Comparing flight and ground groups, the increased cellularity reached statistical significance in the bFGF-treated sponges and showed the same trend with PDGF-BB. When the DNA contents of all flight sponges, irrespective of treatment, were compared to their ground counterparts, there was a highly significant decline in the cellularity of granulation tissue (P<0.005, ANOVA).

The sponge model is useful because there is a characteristic, progressive increase in collagen content until about day 14, within the time frame of the spaceflight experiment. Collagen accumulation was evaluated in these same sets of wound tissues, revealing that the spaceflight environment significantly reduced wound collagen concentration irrespective of the presence or absence of growth factor (Fig. 2 ). The average of all flight vs. ground control sponges showed a highly significant decrease in percent collagen (P<0.01, ANOVA) in (all) flight sponges. Basic FGF release brought about a slight decrease in collagen content of ground controls, consistent with the ability of this cytokine to stimulate expression of metalloproteinases.(19) Collagen concentration of granulation tissue in flight animals treated withbFGF was significantly less than PDGF, but not significantly lower than the placebo treated wounds.

View larger version (44K): [in this window] [in a new window]   Figure 2. Collagen concentration of sponge granulation tissue is diminished in spaceflight. Study groups are the same as depicted in Fig. 1 . Collagen content was determined by amino acid analysis. Collagen concentration (%) determination assumed that the hydroxyproline content in collagen of all groups was not significantly different. When data from all treatment groups were pooled (total), ANOVA showed a highly significant decrease in the collagen concentration of sponges in animals that healed while in orbital flight in the space shuttle (P<0.01). When data were segregated into treatment groups (factor), the decrease in collagen concentration attributable to orbital flight was significant, irrespective of treatment. Although FGF and PDGF treatments were not significantly different from placebo, in the ground group they differed significantly from each other (P<0.05).

This experiment establishes that, at least in young rats, a highly standardized wound repair process is significantly altered at the biochemical level by the spaceflight environment. Irrespective of the presence or absence of a cytokine stimulus, there was a significant inhibition of matrix formation in flight animals. Though basal cellularity was equivalent, stimulated proliferation and immigration of cells into the wound was absent in orbital flight. Previous studies of bone healing in spaceflight have shown small differences in collagen content and some alteration in collagen cross-linking profiles (20) . Although wound collagen content was markedly reduced in orbital flight, there was not a dramatic effect on the overall organization of granulation tissue, confirming that the lack of a gravitational field did not influence mechanical forces that arise in a subcutaneous tissue compartment. Conclusions may be substantially different in other tissues of the musculoskeletal system, where mechanical load plays an important role in stimulating repair and organizing new tissue growth. The observation that there was a blunted response to growth factor stimulation, particularly with respect to cellular influx, suggests that tissue responsiveness is also muted in this experimental situation.

A number of potential mechanisms can be invoked to explain these findings. Effects of spaceflight on bone formation and bone loss are well documented, and some data support the concept that monocyte abundance and leukopoiesis are altered by spaceflight exposure 21-25) . Wound healing is highly dependent on circulating leukocytes for proper development. In particular, the abundance of monocytes, precursors of macrophages, appears to be a rate-limiting parameter in tissue repair (26) . It is possible, therefore, that microgravity has altered the capacity to generate adequate macrophage populations. Stress-induced glucocorticoids might have been a contributing factor. However, corticosteroid levels were equally elevated in all test populations at the time of death (data not shown). An alternative interpretation of the experiment may pertain to fluid dynamics in microgravity. The wound site is characterized by a lack of lymphatics and rapidly generating microvasculature. Microgravity may alter blood flow in or around the wound site and thereby diminish repair capacity. Finally, these experiments do not fully distinguish the effects of microgravity from those other aspects of the space environment. In a previous microgravity wound healing experiment, rats were flown in Cosmos 1884. Although minor differences were noted in connective tissues, the conclusions of the experiment were marred by a 2-day delay in retrieving of the unmanned capsule (27) . The present experimental design did not distinguish between possible effects of radiation and microgravity in orbital flight. A shuttle-based, 1 x g centrifuge study would help to distinguish between these two aspects of the environment.

As it is highly likely that trauma, and perhaps even elective surgery, will take place in the space environment, we think these studies are useful by suggesting that there may be intrinsic defects in the capacity of the body to form new connective tissue. At least one of the potential cellular mechanisms for this response is reduced activation by growth factors known to be present at the wound site. Optimal wound healing during spaceflight may require special procedures.

	ACKNOWLEDGMENTS

 
Supported in part by a grant from the National Institute on Aging and the Department of Veterans Affairs (J.M.D.). We thank M. G. Giro for editorial assistance.

	FOOTNOTES

 
¹ Correspondence: Department of Pathology C-3321 MCN, Vanderbilt University School of Medicine, 21st and Garland Sts., Nashville, TN 37232-2561, USA. E-mail:jeff.davidson{at}vanderbilt.edu

² Current address: BioTech Express, St. Bernard, OH 45217, USA.

³ Abbreviations: AEM, animal enclosure modules; ANOVA, analysis of variance; bFGF, basic fibroblast growth factor; PDGF-BB, platelet-derived growth factor.

Received for publication June 11, 1998. Accepted for publication October 27, 1998.

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