Bacterial Degradation
of Growth Factors

Wyatt G. Payne, MD*†

Terry E. Wright, MD*†

Francis Ko, BS*

Chad Wheeler, BS*†

Xue Wang, PhD*

Martin C. Robson, MD*†

 

*Institute for Tissue Regeneration, Repair, and Rehabilitation, Department of Veteran’s Affairs Medical Center, Bay Pines, Florida.

†The Department of Surgery, University of South Florida, Tampa, Florida.

 

Presented in part at the Wound Healing Society Meeting, May 17, 2001, Albuquerque, New Mexico,

and John A. Boswick, MD, Symposium for Burn and Wound Care, February 18, 2002, Maui, Hawaii.

 

Key words: Growth factors, wound healing, bacterial degradation,
matrix metalloproteinases 

ABSTRACT

Application of exogenous growth factors has been shown to overcome the inhibition of wound healing by bacteria. However, it requires much larger doses of the growth factors than would have been predicted by in vitro studies. It has been suggested that bacterial enzymes such as proteases or the production of matrix metalloproteinases (MMPs) from the interaction of bacteria and tissue may cleave molecules and cause degradation of the growth factors.

Methods: In part 1, 5 million organisms of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli.), Staphylococcus aureus (S. aureus), or Streptococcus faecalis (S. faecalis) were mixed with growth factor basic fibroblast growth factor (bFGF), granulocyte-macrophage colony stimulating factor (GM-b2CSF), transforming growth factor (TGF-b2), or keratinocyte growth factor (KGF-2). The mixtures were incubated at 37˚C and growth factor without bacteria served as a control. Significant bacterial degradation of all growth factors was noted. In part 2, the experimental design was repeated in the presence of confluent fibroblasts. Further degradation in the presence of fibroblasts beyond that of samples with bacteria alone were observed.

Conclusion: Growth factors are degraded in the presence of significant quantities of bacteria. When bacteria are in the presence of soft tissue cells such as fibroblasts, growth factors are further degraded, possibly due to bacterial proteases or MMPs. These data support the need for control of bacterial burden before application of exogenous growth factors in contaminated wounds in the clinical setting.

INTRODUCTION

Growth factors and cytokines are the mediators for the various processes of wound healing.1–6 Many growth factors have been shown to affect the processes of wound healing, and the list continues to grow. Bacteria have been shown to affect all processes of wound healing.7 This may be due to a bacterial effect on growth factor function, either by direct inhibition of function or by diminishing the growth factor quantity itself.8–11

In the rat model of contaminated wound contraction, it required at least 100 micrograms/cm2 of basic fibroblast growth factor (bFGF) in a saline vehicle to overcome the inhibition to wound contraction caused by more than 105 bacteria per gram of tissue.12 This could be reduced 10- to 100-fold by changing the vehicle to carboxymethylcellulose.13 The carboxymethylcellulose was thought to protect the growth factor from bacterial degradation. In clinical trials of exogenous growth factors, our center has shown positive effects from growth factor therapy,14–17 but other centers have mixed results.3 However, we have always prepared the wounds before treatment with exogenous growth factors by obtaining bacterial balance measured by wound tissue biopsies showing 1 x 106 or fewer bacteria per gram of tissue.10 This was not uniformly the case at other centers.3

Tarnuzzer and Schultz5 showed that high levels of bacteria led to up-regulated inflammatory mediators, up-regulated matrix metalloproteinases (MMPs), and decreased levels of endogenous growth factors in chronic wounds. These observations have led investigators in recent clinical trials involving growth factors to recommend control of bacterial burden of a wound before treatment with exogenous growth factors.18

The purpose of the present experiment was to demonstrate the effects of bacteria on various cytokine growth factors in culture alone and when tissue components (fibroblasts) are added to the culture.

METHODS

The experiment was divided into 2 parts. In part 1, 5 x 106 organisms from an 18-hour broth culture of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Streptococcus faecalis were mixed with 500 pg/mL of bFGF, granulocyte-macrophage colony stimulating factor (GM-CSF), keratinocyte growth factor - 2 (KGF-2), or transforming growth factor b2 (TGF-b2). Growth factor without bacteria was used as a control. The mixtures were incubated at 37˚C in a 5% carbon dioxide incubator at pH 7.4. Aliquot samples were taken at 0, 3, 6, and 24 hours and analyzed for quantity of growth factor by ELISA technique.

In part 2, the experimental design was repeated, again using growth factor without bacteria as control. However, confluent fibroblasts were added to the bacterial laden incubation mixture to determine their effect. Again aliquot samples were taken at 0, 3, 6, and 24 hours, and ELISA analysis for growth factor quantity was performed.

Chi-square analysis was used to compare cytokine growth factor degradation in bacterial environment at 24 hours to degradation in culture media alone at 24 hours. Similarly, chi-square analysis compared degradation at 24 hours when fibroblasts were added to the bacteria-cytokine growth factor cultures to that in the bacteria-cytokine growth factor cultures without fibroblasts at 24 hours.

RESULTS

In part 1, all 4 species of bacteria significantly degraded each of the 4 growth factors by 24 hours of exposure (P < 0.05) (Tables 1 through 4). Basic FGF was degraded from 37% to 76.6% by bacteria versus only 12% when no bacteria was present (control; P < 0.05; Table 1). GM-CSF was degraded from 39.4% to 48.6% by bacteria versus only 16% when cultured without bacteria (control; P < 0.05; Table 2). TGF-b2 was degraded from 42.1% to 55.4% by the various strains of bacteria compared with 12% for the control (P < 0.05; Table 3). KGF-2 was degraded 45% to 63.3% by bacteria compared with 15.1% when no bacteria were present (control; P < 0.05; Table 4).

In part 2, adding tissue cells (fibroblasts) to the growth factor-bacteria cultures tended to accentuate the action of the bacteria (Tables 5 through 8). The increase in percent degradation ranged up to 23%. Adding fibroblasts increased degradation of b-FGF by 2 of the 4 bacterial species (Table 5). This increase was statistically significant for P. aeruginosa (P < 0.05). The most marked increase in degradation occurred to GM-CSF (Table 2, Table 6). Three of the bacterial species’ degradation were significantly enhanced with fibroblasts (P < 0.05). There were trends for TGF-b2 to be degraded further with addition of fibroblasts to the bacterial cultures in 3 of 4 of the species (Table 3, Table 7). However, these increases were not statistically significant (P > 0.05). The least differences were seen with KGF-2 (Table 4, Table 8). None of the differences were of statistical significance (P > 0.05).

DISCUSSION

Growth factors regulate and coordinate the processes of wound healing.1,2 Disruption of the process leads to a chronic non-healing wound.19 High bacterial levels can interfere with the orderly progression of wound healing and interrupt the process because of the effects on endogenous growth factors.5,7,10,18,20 In chronic wounds, repetitive trauma, ischemia, and infection increase proinflammatory cytokines, increase MMPs, and decrease the levels of growth factors.4,5,9,21 Tissue repair is interrupted, and failure of the wound to heal occurs, resulting in a chronic wound.

The results of this study confirm degradation of growth factor levels in the presence of significant quantities of bacteria in a controlled experimental environment. Decrease in growth factor levels up to 69% were noted for gram-negative species and to 76% for gram-positive species (Table 1).

Further enhancement of bacterial degradation of the growth factors occurred in the presence of fibroblasts. The presence of fibroblasts caused further growth factor degradation by up to 33% (Tables 2 and 6). Fibroblasts were chosen because in culture they provide a useful model for wound contraction22 and because of their role in production of MMPs.5 It is likely that bacteria alone secrete proteases, which, by cleaving complex molecules, can degrade and inactivate growth factors. When bacteria and fibroblasts are present together, enhanced degradation occurs, due possibly to a synergistic effect of bacteria on fibroblasts or MMPs. Our study implicates bacteria as the causative factor and fibroblasts as an affected enhancer of growth factor degradation.

Treatment of wounds with the array of growth factors tested in this study have all been shown to clinically improve chronic wound healing to some extent.14–17,23 These trials were performed with attention to control of bacterial burden, which may be a decisive factor in improving the utility of growth factor therapy in chronic wound care.

Control of bacterial burden should be accomplished before use of exogenously applied growth factors to avoid degradation in the chronic wound environment.

Conclusions

Significant amounts of bacteria that cause invasive infection (> 105 organisms per gram of tissue) cause degradation of growth factors in vitro. The presence of tissue components (fibroblasts) enhances and increases this degradation process. Though the exact cause is not known, bacterial proteases and MMPs have been shown to degrade growth factors in wound tissue, and this mechanism could account for the degradation of bFGF, GM-CSF, KGF-2, and TGF-b2, seen in this study. We have demonstrated degradation of these growth factors in the presence of bacteria alone. The further enhanced degradation of growth factors observed with mixtures of bacteria and fibroblasts implicate a synergistic effect caused by this interaction. The mechanism is not fully understood. Further experimentation will be necessary to identify the exact cause.

REFERENCES

1. Robson MC: The role of growth factors in the healing of chronic wounds. Wound Rep Regen  5:12–17, 1997.

2. Stadelmann WK, Digenis AG, Tobin GR: Physiology and healing dynamics of chronic cutaneous wounds. Am J Surg 176(Suppl 2A):26S–38S, 1998.

3. Robson MC, Mustoe TA, Hunt TK: The future of recombinant growth factors in wound healing. Am J Surg 176(Suppl 2A):80S–82S, 1998.

4. Schultz GS, Mast BA: Molecular analysis of the environment of healing and chronic wounds: Cytokines, proteases, and growth factors. Wounds 10(Suppl F):1F–9F, 1998.

5. Tarnuzzer RW, Schultz GS: Biochemical analysis of acute and chronic wound environments. Wound Rep Regen 4:321–325, 1996.

6. Robson MC: Exogenous growth factor application effect on human wound healing. Prog Derm 30:1–6, 1996.

7. Robson MC, Stenberg BD, Heggers JP: Wound healing alterations caused by infection. Clin Plast Surg 17:485–492, 1990.

8. Robson MC: Treating bacterial infections in chronic wounds. Contemp Surg Suppl:9–11, 2000.

9. Yager DR, Chen SM, Ward SI, et al: Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Rep Regen 5:23–32, 1997.

10. Robson MC: Wound infection: A failure of wound healing caused by an imbalance of bacteria. Surg Clin North Am 77:37–650, 1997.

11. Stadelmann WK, Digenis AG, Tobin GR: Impediments to wound healing. Am J Surg 176 (Suppl 2A):39S–47S, 1998.

12. Hayward P, Hokanson J, Heggers J, et al: Fibroblast growth factor reverses the bacterial retardation of wound cantraction. Am J Surg 163:288–293, 1992.

13. Kuhn MA, Page L, Nguyen K, et al: Basic fibroblast growth factor in a carboxymethylcellulose vehicle reverses the bacterial retardation of wound contraction. Wounds  13:73–80, 2001.

14. Robson MC, Hill DP, Smith PD, et al: Sequential cytokine therapy for pressure ulcers; clinical and mechanistic responses. Ann Surg 231:600–611, 2000.

15. Robson MC, Phillips LG, Cooper DM, et al: Safety and effect of transforming growth factor beta–2 for treatment of venous stasis ulcers. Wound Rep Regen 3:157–167, 1995.

16. Robson MC, Phillips LG, Thomason A, et al: Recombinant human platelet-derived growth factor-bb for the treatment of chronic pressure ulcers. Ann Plast Surg 29:193–201,1992.

17. Robson MC, Phillips LG, Lawrence WT, et al: The safety and effect of topically applied recombinant basic fibroblast growth factor on the healing of chronic pressure sores. Ann Surg 216(4):401–408, 1992.

18. Robson MC, Smith PD: Topical use of growth factors to enhance healing, in Cutaneous Wound Healing. Falanga V, ed. London: Martin Dunitz Limited, 2001:379–398.

19. Lazarus GS, Cooper DM, Knighton DR, et al: Definitions and guidelines for assessment of wounds and evaluation of healing. Arch Derm 130:489–493, 1994.

20. Bennett NF, Schultz GS: Growth factors and wound healing: Part II. Role in normal and chronic wound healing. Am J Surg 166:74–81, 1993.

21. Mast BA, Schultz GS: Interactions of cytokines, growth factors, and proteases in acute and chronic wounds. Wound Rep Regen  4:411–420, 1996.

22. VandeBerg JD, Rudolph R: Cultured myofibroblasts: A useful model to study wound contraction and pathological contracture. Ann Plast Surg 2:111–120, 1985.

23. Robson MC, Phillips TJ, Falanga V, et al: Randomized trial of topically applied repifermin (recombinant human keratinocyte growth factor-2) to accelerate wound healing in venous ulcers. Wound Rep Regen 9:347–352, 2001.

 

Table 1. Basic Fibroblast Growth Factor (bFGF) Degradation by Bacteria

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          5.2%                 24.4%                  67.7%                    22.7%                     22.7%

6                          7.2%                 35.1%                  43.9%                    40.6%                     30.2%

24                        12%                   37%*                   69%*                     76.6%*                    73.6%*

 

* P < 0.05.

 

Table 2. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Degradation by Bacteria

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          9.4%                  5.5%                    12%                        5%                        14.5%

6                          14%                  36.6%                  32.8%                    30.2%                     34.8%

24                        16%                 39.4%*                34.7%*                   48.6%*                    37.3%*

 

* P < 0.05.

 

Table 3. Transforming Growth Factor b2 (TGF b2) Degradation by Bacteria

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          9.7%                   30%                   29.4%                    34.2%                     33.3%

6                         10.0%                  50%                   46.3%                    39.3%                      39%

24                        12%                 54.1%*                55.4%*                   42.1%*                    45.4%*

 

* P < 0.05.

 

Table 4. Keratinocyte Growth Factor-2 (KGF-2) Degradation by Bacteria

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          8.3%                  8.4%                   14.7%                     7.2%                       4.6%

6                          8.8%                 31.4%                  33.5%                    20.7%                      23%

24                       15.1%                63.3%*                53.4%*                   50.6%*                     45%*

 

* P < 0.05.

Table 5. Basic Fibroblast Growth Factor (bFGF) Degradation by Bacteria in Presence of Fibroblasts

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          6.2%                 38.5%                  25.3%                    32.8%                      64%

6                          9.7%                 46.9%                  40.8%                    45.9%                     74.1%

24                        20%                 59.7%*                  69%*                     76.6%*                    86.6%*

* P < 0.05.

 

Table 6. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Degradation by Bacteria in Presence of Fibroblasts

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          7.9%                   19%                   14.7%                    15.8%                      20%

6                         13.7%                47.8%                  36.4%                    27.7%                     37.3%

24                       16.5%                 73%*                  57.3%*                   60.9%*                    48.5%*

 

* P < 0.05.

 

Table 7. Transforming Growth Factor Beta-2 (TGF beta-2) Degradation by Bacteria in Presence of Fibroblasts

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          4.4%                   53%                   55.8%                      44%                       49.3%

6                         14.7%                53.4%                  58.7%                    45.4%                     54.7%

24                       15.7%                53.8%*                60.9%*                   55.2%*                    54.7%*

 

* P < 0.05.

 

Table 8. Keratinocyte Growth Factor-2 (KGF-2) Degradation by Bacteria in Presence of Fibroblasts

 

                                               Pseudomonas       Escherichia        Staphylococcus       Streptococcus            

Time (h)            Control            aeruginosa                coli                      aureus                   faecalis

 

0                           0%                     0%                       0%                         0%                          0%

3                          7.8%                 17.1%                  18.6%                     5.2%                      27.2%

6                          8.8%                 22.1%                  38.5%                    10.7%                      35%

24                       16.3%                76.6%*                55.1%*                   47.8%*                    51.6%*

 

* P < 0.05.