Wound Healing and the Immune System

Thought initially to be the hallmark of the inflammatory phase, a large cellular infiltrate is now known not to be beneficial to the rate of wound closure. Neutrophil-depleted mice demonstrate markedly faster wound closures than their wild-type counterparts. Also, mice deficient in the tumor necrosis factor receptor p55 have a reduced leukocyte infiltration and improved wound closure. Mice deficient in TGF-b signaling protein SMAD3 have reduced monocytic infiltration, reduced matrix, and an increased rate of wound closure [7]. Similarly, estrogen has shown positive effects on cutaneous wound healing by downregulating the macrophage migration inhibitory factor (MIF) and decreasing inflammation. MIF knockout animals also have fast wound closure, decreased inflammatory cell infiltrate, and increased matrix deposition. All these studies indicate that reduced cellular infiltration is associated with a faster wound healing phenotype. It is, however, important to determine whether faster healing is necessarily better healing, as the hastily healed wound of SMAD3-deficient animals might lack resilience to breaking forces.

Whereas a large neutrophilic infiltrate adversely affects wound closure rates, other cell types are also important in positively or negatively modifying the wound-healing process. g-S T cells are a subset of the T-cell population. Unlike the majority of the T-cells, which are of an a-b subtype and involved in cellular and humoral immunity, these cells reside mostly in epithelial tissue. previously thought to be unimportant, they are now known to proliferate in response to injury. When stimulated, they produce FGF-7 and KGF-2 (FGF-10). Animals deficient in g-S T-cells exhibit delayed wound closure, which can be rescued with the application of either g-S T cells or KGF [8]. Thus, this T-cell subtype and its products serve to enhance wound closure. The results of similar studies in T-cell deficient nude mice are less clear. Wound strength was increased in the first 2 weeks post injury, but decreased below that of wild-type

Table II Summary of All Clinical Trials Pertaining to the Use of Growth Factors in Wound Healing.

Druga

Year

Type

No.

Outcome

For chronic diabetic neuropathic ulcers PDGF-ßß

III

250

No difference between good ulcer care alone and 100 |lg/g PDGF

PDGF-ßß

III

172

Worse healing in PDGF group in comparison to good ulcer care alone

PDGF-ßß

III

382

Improved healing only in high-dose 100 | g/g group

PDGF-ßß

1996

III

118

Improved healing in PDGF 30 | g/g group

FGF

1995

II

17

No difference

For chronic nonhealing venous ulcers rhKGF-2 2001

II

94

More patients achieve 75% wound closure in KGF-2 treated group

Epithelial cultures

2002

I

11

7 patients healed, 4 did not

PR

2000

II

86

No difference after biweekly prescription for 9 months

PR

1999

II

15

No significant difference

GM-CSF

1999

I

38

Complete healing seen in 90.4% of patients without recurrence

GM-CSF

1999

II

60

Improvement of number of animals with complete healing by 13 weeks

EGF

1992

II

35

Improvement in rate of complete healing

For chronic pressure ulcers TGF-b3

2001

II

270

High dose (2.5 ||g/cm2) TGF-b3 healed faster on fourth visit but no difference at the end

GM-CSF and/or FGF

2000

II

FGF alone achieved the fast rate of wound closure

PDGF-bb

1999

II

124

Significant increase in wounds achieving > 90% healing

PDGF-bb

1994

II

41

Effective in decreasing wound area after 28 days

PDGF-bb

1992

I/II

20

Significant improvement in closure

For miscellaneous wounds PDGF-bb

2002

II

21

Abdominal wound separation Decreased in time to complete healing

GM-CSF

2002

I

5

Chronic leg ulcers Appears effective

GH

2002

II

24

Donor site healing in burned No improvement in donor site healing time adults

Bovine basic

2000

II

1024

Burn wounds, donor sites, Treatment arm (bFGF) improved wound

GM-CSF

GM-CSF GM-CSF EGF

GM-CSF PR

EGF PR

2001

2000

1998

998 997 995

992 991

991 990

1989

Ia II Ia

II II

II II

Ia and chronic dermal ulcers

7 4-mm full-thickness punch biopsy

134 Chronic lower extremity diabetic ulcers

29 Chronic refractory wounds

10 5-mm punch wound

16 Chronic leg ulcers

17 Bilateral split-thickness skin graft donor site

Chronic nonhealing wounds

13 Chronic diabetic foot ulcers

> 8 weeks 35 Leprosy wounds

18 Nonhealing lower extremity wounds

9 Chronic wounds > 12 m

32 Chronic nonhealing wounds

12 Donor sites closure with superficial second best

Daily PDGF treatment significantly improved closure rate

57.5% achieved complete healing in 20 weeks with 21% recurrence

One third healed completely in 6 weeks and another 11 decreased in size by 50% Did not show improvement in healing time Nonsignificant difference No difference

Wounds of > 75 weeks old reepithelialized in 10 weeks posttreatment Results in much faster healing than control

Rapid filling of treated wounds No improvement in healing rate

All wounds heal in 34 days Significantly more wounds in intervention wound achieving epithelialization Improvement in epithelialization by 1—1.5 days on average a Abbreviations: PDGF-bb, platelet-derived growth factor-bb; FGF, fibroblast growth factor; rhKGF-2, recombinant human keratinocyte growth factor; PR, platelet releasates; GM-CSF, granulocyte/macrophage colony stimulating factor; EGF, epidermal growth factor; TGF-b3, transforming growth factor b3; GH, growth hormone.

mice after 6 weeks, indicating that T cells can have both a stimulatory and inhibitory effect.

Clinical Trials in Wound Healing Using Growth Factors

Currently, PDGF is the only growth factor approved by the FDA for clinical use in the treatment of chronic wounds. This undertaking, however, took pooled data from four Phase III clinical studies treating neuropathic ulcers in almost 1,000 diabetic patients [9]. The variability seen in the four studies underlines the difficulty in performing wound healing clinical trials. With all the trials taken together, PDGF gave a modest 10 percent increase in the overall rate of complete wound healing. Other ulcers that could potentially benefit from its use include chronic pressure ulcers that showed promise among three Phase II trials totaling 175 patients. However, Phase III studies led to no improvement.

Even though only one growth factor has been approved clinically for chronic wounds, many studies on growth factors have been done. A summary of clinical trials performed categorized by indication is given in Table II. Many drugs show early promise in Phase I studies but fail to show efficacy in blinded studies performed in multiple institutions. Alternatively, the improvement in healing is so slight that it does not achieve clinical or statistical significance. Such small differences in healing rates would not justify the expense needed for the production of the drug. Difficulty in these trials also can be attributed to confounders such as small size (many trials contain fewer than 20 patients), patient noncompliance (i.e., pressure sores), and rapid degradation of the growth factors.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a growth factor that has shown early clinical promise in the treatment of chronic nonhealing venous ulcers in numerous phase I and phase II studies. Similarly, recombinant keratinocyte growth factor (KGF) has shown success in a Phase II study involving 94 patients with chronic venous ulcers. In the treated group, more patients achieved 75 percent wound closure than in the placebo group. The next few years will tell whether their efficacy persists in Phase III studies.

Future Directions: Involvement of the Nervous System

Whereas significant wound healing research in the past has focused on the role of circulating humoral growth factors, evidence now points to the nervous system as being central in cutaneous healing. It first came from observations that poorly innervated areas of the body are more resistant to wound healing. Diabetic patients, with associated peripheral neuropathies, suffer from poorly healing extremities. They are 15 to 20 times more likely to undergo amputations than their nonneuropathic diabetic counterparts. Although there could be many contributory factors, impaired healing in diabetic wounds has been postulated to be a result of their reduced levels of substance P, a proinflammatory cytokine released by nerve endings after wounding to recruit cells and promote vasodilatation. Because of the diabetic patients' increased levels of neutral endopeptidases, substance P is degraded more rapidly. Indeed, topical application of substance P or endopeptidase inhibitors have resulted in better wound healing. Similarly, mice lacking the low-affinity NGF receptor p75 demonstrate impaired healing while the topical application of a nerve growth factor to their wounds improved healing [10].

Vacuum-Assisted Wound Closure

Although significant progress in wound healing has focused on the role of growth factors, many studies have highlighted the success of an emerging modality that applies subatmospheric pressures to a wound. The VAC (Kinetic Concepts Inc.) is one such device that utilizes a modified dressing consisting of a porous sponge with an inlaid suction tubing that is secured to the wound using an occlusive dressing [10]. An attached pump maintains a continuous negatively pressured wound environment while removing edematous fluid. In addition to decreasing tissue edema and bacterial load, it has been hypothesized that this device also applies micromechanical forces to the wound stretching individual cells, thereby promoting proliferation in the wound micro-environment. The VAC has been associated with accelerated development of granulation tissue, early reepithelialization, and faster healing of both burn and complex wounds. Although many studies are underway to understand the physiological effects of this device, several clinical studies have clearly found that this treatment facilitates wound healing.

Conclusion

Cutaneous wound healing is a potpourri of humoral and cellular factors. Although great strides have been made in the treatment of chronic wounds through the control of infection as well as nutritional supplementation, this chapter is clearly incomplete. The next stride will hinge on finding an optimal combination of growth factors and defining other factors that lead to effective wound healing such as micro-mechanical forces. Any newly developed treatment options have to be both efficacious and cost-effective. Only when such options are available will the surging tide of poorly healed chronic wounds ebb.

Glossary

Chronic wounds: Open skin surfaces that fail to close after normal expected time for wound closure. Common causes include metabolic dis orders such as malnutrition, diabetes, and steroid dependence; or mechanical causes such as pressure, venous congestion, arterial occlusion, and after radiation or burns.

Micromechanical forces: Tension, surface tension, shear, compression, and gravity are all examples of micromechanical forces. It is known that cellular morphology can directly influence cellular function, growth, and differentiation. Lack of micromechanical forces can result in apoptosis. Conversely, application of these micromechanical forces to wounds has been associated with improved cell growth and improved wound healing.

Reepithelialization: The migration of keratinocytes to seal an open wound, considered by many to be a good predictor of successful wound healing.

References

1. Werner, S., and Grose, R. (2003). Regulation of wound healing by growth factors and cytokines. Physiol. Rev. 83(3), 835-870. This review summarizes the results of expression studies that have been performed in rodents, pigs, and humans to localize growth factors and their receptors in skin wounds.

2. LeGrand, E. K. (1998). Preclinical promise of becaplermin (rhPDGF-bb) in wound healing. Am. J. Surg. 176(2A Suppl), 48S-54S. This article reviews how the study of rhPDGF-bb in animal wound healing models has assisted in addressing the potential clinical utility of rhPDGF-bb.

3. Joris, I., Braunstein, P. W., Jr., Pechet, L., and Majno, G. (1980). Effect of thrombocytopenia on wound healing. A study in the rat. Exp. Mol. Pathol. 33(3), 283-291. The addition of a neutralizing anti-platelet antibody to mice has surprisingly led to no change in the wound healing rates, but significantly increased the number of macrophages and T-cells present in the wound.

4. Martin, P. (1997). Wound healing—aiming for perfect skin regeneration. Science 276(5309), 75-81. A review on wound healing focusing on the contributing cell types during the phases of proliferation, migration, matrix synthesis, and contraction, as well as the growth factor and matrix signals present at a wound site.

5. Livant, D. L., Brabec, R. K., Kurachi, K., Allen, D. L., Wu, Y., Hoaseth, R., Andrews, P., Ethier, S. P., and Markwart, S. (2000). The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice. J. Clin. Invest. 105(11), 1537-1545. The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to migrate and also stimulates reepithelialization and contraction of dermal wounds in healing-impaired, obese diabetic mice.

6. Kirchner, L. M., Meerbaum, S. O., Gruber, B. S., Knoll, A. K., Bulgrin, J., Taylor, R. A., and Schmidt, S. P. (2003). Effects of vascular endothe-lial growth factor on wound closure rates in the genetically diabetic mouse model. Wound Repair Regen. 11(2), 127-131. Topically applied vascular endothelial growth factor improves wound closure rates in diabetic animals in a full-thickness wound model in genetically diabetic mice.

7. Ashcroft, G. S., Yang, X., Anzano, M., Green-Wild, T., Wahl, S. M., Glick, A. B., Weinstein, M., Letterio, J. L., Mizel, D. E., Deng, C., and Roberts, A. B. (1999). Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat. Cell Biol. 1(5), 260-266. In contrast to predictions made on the basis of the ability of exogenous TGF-0 to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by increased reepithelialization and reduced local infiltration of monocytes.

8. Jameson, J., Ugarte, K., Chen, N., Yachi, P., Fuchs, E., Biosmenn, R., and Hauran, W. L. (2002). A role for skin gS T cells in wound repair. Science 296(5568), 747-749. Activated DETCs produce keratinocyte growth factors (KGFs) and chemokines, raising the possibility that DETCs play a role in tissue repair. We performed wound healing studies and found defects in keratinocyte proliferation and tissue reepithe-lialization in the absence of wild-type DETCs.

9. Wieman, T. J. (1998). Clinical efficacy of becaplermin (rhPDGF-bb) gel. Becaplermin Gel Studies Group. Am. J. Surg. 176(2A Suppl), 74S-79S. The results of four multicenter, randomized, placebo-controlled, parallel group studies of the efficacy of becaplermin gel are reviewed here.

10. Gibran, N. S., Jang, Y. C., Isik, F. F., Greenhalgh, D. G., Muffley, L. A., Underwood, R. A., Uswi, M. L., Larsen, J., Smith, D. G., Bunett, N., Ansel, J. C., and Olarnd, J. E. (2002). Diminished neuropeptide levels contribute to the impaired cutaneous healing response associated with diabetes mellitus. J. Surg. Res. 108(1), 122-128. We demonstrated fewer nerves in the epidermis and papillary dermis of skin from human subjects with diabetes. Likewise, db/db murine skin had significantly fewer epidermal nerves than nondiabetic littermates. The use of substance P in improving wound healing is substantiated by a murine model of impaired healing.

Capsule Biography

Dr. Chan is a surgical resident at the Brigham and Women's Hospital and a research fellow in the Tissue Engineering and Wound Healing Laboratory in the Department of Plastic Surgery at Harvard Medical School.

Dr. Liu is a surgical resident at Our Lady of Mercy Medical Center in The Bronx, New York. He is taking time off from residency to work as a research fellow at the Tissue Engineering and Wound Healing Laboratory of Brigham and Women's Hospital/Harvard Medical School. His current research, supported in part by a grant from the Center for Integration of Medicine & Innovative Technology, focuses on the use of micro-mechanical forces, platelet releasates, and stem cells to enhance wound healing.

Dr. Orgill is an Associate Professor of Surgery at Brigham & Women's Hospital/Harvard Medical School, Associate Chief of the Division of Plastic Surgery, and Associate Chief of the Burn Unit. His Tissue Engineering and Wound Healing Laboratory is currently investigating the use of micro-mechanical forces in wound healing, the use of melanocytes to correct skin and hair pigment disorders, and the engineering of durable tissue substitutes for repairing tissue defects. His research is supported by BWH and the Center for Integration of Medicine & Innovative Technology.

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