Clinical application of growth factors and cytokines in wound healing

Wound healing is a complex and dynamic biological process that involves the coordinated efforts of multiple cell types and is executed and regulated by numerous growth factors and cytokines. There has been a drive in the past two decades to study the therapeutic effects of various growth factors in the clinical management of nonhealing wounds (e.g., pressure ulcers, chronic venous ulcers, diabetic foot ulcers). For this review, we conducted an online search of Medline/PubMed and critically analyzed the literature regarding the role of growth factors and cytokines in the management of these wounds. We focused on currently approved therapies, emerging therapies, and future research possibilities. In this review, we discuss four growth factors and cytokines currently being used on and off label for the healing of wounds. These include granulocyte‐macrophage colony‐stimulating factor, platelet‐derived growth factor, vascular endothelial growth factor, and basic fibroblast growth factor. While the clinical results of using growth factors and cytokines are encouraging, many studies involved a small sample size and are disparate in measured endpoints. Therefore, further research is required to provide definitive evidence of efficacy.     

Human plasma fibronectin potentiates the mitogenic activity of platelet-derived growth factor and complements its wound healing effects

Integrin-mediated cell adhesion and growth factor stimuli are both required for optimal control of cell proliferation. In the context of skin injury, cell-derived fibronectin and platelet-derived growth factor play important roles in the stimulation of cell proliferation and migration, activities that are crucial to the healing process. To assess the ability of exogenously supplied plasma-derived fibronectin to stimulate wound repair and to study its ability to cooperate with platelet-derived growth factor-BB during healing, we devised a novel topical delivery formulation that allows the controlled release of both molecules to a wound. Using this topical formulation and the rabbit ear model of dermal wound healing, we show that plasma fibronectin is a potent stimulator of the wound healing process. We also show that administration of fibronectin and platelet-derived growth factor-BB in combination has additive wound healing effects. Finally, we report novel findings on the ability of soluble plasma fibronectin to potentiate the mitogenic effects of platelet-derived growth factor-BB in vitro. These findings not only show that optimal concentrations of exogenous fibronectin administered using an effective delivery system stimulate wound healing; they also suggest that PDGF-BB should be administered with fibronectin to achieve optimal therapeutic stimulation of wound healing.     

Therapeutic role of growth factors in treating diabetic wound

Wounds in diabetic patients, especially diabetic foot ulcers, are more difficult to heal compared with normal wounds and can easily deteriorate, leading to amputation. Common treatments cannot heal diabetic wounds or control their many complications. Growth factors are found to play important roles in regulating complex diabetic wound healing. Different growth factors such as transforming growth factor beta 1, insulin-like growth factor, and vascular endothelial growth factor play different role...     

Selective release of cytokines,chemokines, and growth factors by minced skin in vitro supports the effectiveness of autologous minced micrografts technique for chronic ulcer repair

A new effective surgical procedure to repair chronic ulcers called minced micrografts technique has been recently reported. The technique consists in spreading a finely minced skin sample upon the wound bed. In this study, we investigate the in vitro release of cytokines (interleukin‐6, tumor necrosis factor‐α, interleukin‐1α, and granulocyte‐colony stimulating factor), chemokines (monocyte chemoattractant protein‐1 and growth‐related oncogene‐α), and growth factors (platelet‐derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor, and nerve growth factor) by minced (referred to as the minced sample) vs. not minced (referred to as the whole sample) human skin biopsy samples from the same donor. Factor release in the culture medium at different time points was detected using a multiplexed protein assay. The minced sample, which could behave like the skin fragments used in vivo in the autologous minced micrografts technique, expressed higher levels of tumor necrosis factor‐α, interleukin‐1α, platelet‐derived growth factor, and basic fibroblast growth factor, and lower levels of interleukin‐6, monocyte chemoattractant protein‐1, growth related oncogene‐α, and vascular endothelial growth factor compared with the whole sample. In conclusion, mincing of healthy skin may allow appropriate regulation of the inflammatory phase of wound healing and could induce overexpression of some growth factors, which facilitates the proliferative phase of healing.     

Innovative Therapies in the Treatment of Keloids and Hypertrophic Scars

Keloids and hypertrophic scars are benign fibrous overgrowths of scar tissue, which results from an abnormal response to trauma. Several therapeutic modalities have been described for the treatment and prevention of these conditions, but the optimal management approach has not yet been defined. This article reviews the most recent, innovative, therapeutic strategies for the management of hypertrophic scars and keloids, including mitomycin-C, tamoxifen citrate, methotrexate, imidazolaquinolines, retinoids, calcineurin inhibitors, phenylakylamine calcium channel blockers, botulinum toxin, vascular endothelial growth factor inhibitors, hepatocyte growth factor, basic fibroblast growth factor, interleukin-10, manosa-6-phosphate, transforming growth factor beta, antihistamines, and prostaglandin E2. No consensus in treatment regimens has been reached due to the limited evidence-based information found in the literature. Most therapeutic options have potential effectiveness as both monotherapy and as combination therapy. However, recent reports offer novel modalities that may approach scarring from different angles.Wound healing, as a normal biological process in the human body, is accomplished through different phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal effectively, all four phases must occur in the right sequence. Different factors may interfere with one or more phases of this process. Scars are areas of fibrous tissue that replace normal skin (or other tissue) after injury. A scar results from the biological process of wound repair in the skin and other tissues of the body and constitutes a natural part of the healing process. Scarring is considered abnormal when the amount of fibrosis is excessive or suboptimal, as in hypertrophic, atrophic, or keloidal scars; when it affects normal function; and when it is symptomatic. Scars are also considered abnormal when they are disfiguring or aesthetically distressing to the patient. Several therapeutic modalities have been described for the treatment and prevention of scars, but the optimal management approach has not yet been defined. In this article, the most recent innovative therapeutic strategies for the management of hypertrophic scars and keloids are described. Diverse reports of novel treatments for abnormal scarring from the PubMed/ MEDLINE/Clinical trials.gov database were collected. Most therapeutic options have potential effectiveness as both monotherapy and as combination therapy for the management of abnormal scarring.     

Synergistic actions of platelet-derived growth factor and the insulin-like growth factors in vivo.

Topical application of growth factors has been shown to benefit both normal and impaired wound healing. In normal tissue repair, resident cells produce a "cocktail" of various types of growth factors that overlap in function. In vitro studies have proved that growth factor combinations can act synergistically to enhance cellular function beyond that achieved with individual growth factors. To determine whether similar combinations have a synergistic effect in vivo, we applied growth factor combinations topically to full-thickness skin wounds created in genetically diabetic mice. The C57BL/KsJ-db/db mouse is obese and has insulin-resistant diabetes, and it has been proved that this mouse has markedly impaired wound healing. Topical application of platelet-derived growth factor, insulin-like growth factor-I, or insulin-like growth factor-II enhances healing in this model. Marked synergism was found when platelet-derived growth factor and insulin-like growth factor-II were combined to produce augmentation in wound closure beyond that achieved by application of the individual growth factors. The synergistic effect allowed for improved tissue repair at doses of platelet-derived growth factor and insulin-like growth factor-II that were ineffective when applied individually. The addition of insulin-like growth factor-I or insulin to platelet-derived growth factor produced no significant synergism. Because multiple growth factors are released in the wound during the healing process, it is not surprising that their combination further enhances healing. Growth factor combinations should become an important addition to the armamentarium for the treatment of chronic, nonhealing wounds.     

Wound repair by bone marrow stromal cells through growth factor production

We have previously shown that treatment with bone marrow stromal cells (BMSCs) augments the healing of fascial wounds in the rat. However, the biochemical mechanism by which BMSCs improve wound healing was not investigated. Growth factors have been shown to play a key role in repairing damaged tissue. In this study, we investigated whether BMSCs are capable of producing growth factors that play a critical role in healing of the damaged tissue. Growth factor expression in BMSCs stimulated with pro-inflammatory cytokines or wound superfusate was measured by RT-PCR and growth factor-specific quantitative sandwich enzyme-linked immunosorbent assay (ELISA). RT-PCR analysis demonstrated that BMSCs are capable of expressing transforming growth factor beta-1 (TGF-beta1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) constitutively or upon stimulation with LPS, IL-1alpha, or TNF-alpha. Quantitative analysis of growth factor production by ELISA showed that BMSCs do not secrete TGF-beta1, EGF or VEGF in response to uninjured fascia tissue superfusate; however, production of these growth factors is significantly increased when cells were stimulated with wound tissue superfusate. The ability of wound to stimulate growth factor production in BMSCs could be detected as early as day 1 and lasted through day 7 after wounding. Thus, growth factor production by BMSCs in response to wound microenvionment suggests that BMSCs might augment wound healing through the responsive secretion of growth factors that enhance angiogenesis and promote wound repair.     

Regulatory effects of introduction of an exogenous FGF2 gene on other growth factor genes in a healing tendon

In this study of a tendon injury model, we investigated how injection of a vector incorporating one growth factor gene changes expression levels of multiple growth factor genes in the healing process. The flexor tendon of chicken toes was completely cut and repaired surgically. The tendons in the experimental arm were injected with an adeno‐associated virus‐2 vector incorporating basic fibroblast growth‐factor gene, whereas the tendons in the control arm were not injected or injected with sham vectors. Using real‐time polymerase chain reaction, we found that, within the tendon healing period, a set of growth factor genes—transforming growth factor‐β1, vascular endothelial growth factor, and connective tissue growth factor—were significantly up‐regulated. Expression of the platelet‐derived growth factor‐B gene was not changed, and the insulin‐like growth factor was down‐regulated. A tendon marker gene, scleraxis, was significantly up‐regulated in the period. Our study revealed an intriguing finding that introduction of one growth factor gene in the healing tendon modulated expression of multiple growth factor genes. We believe this study may have significant implications in determining the approach of gene therapy, and the findings substantiate that gene therapy using a single growth factor could affect multiple growth factors.     

Topical insulin in wound healing: a randomised, double-blind, placebo-controlled trial

Two studies were carried out to assess the relative roles of insulin and zinc in the acceleration of wound healing. In the first study, six diabetic and five non-diabetic human volunteers had two uniform cuts created, one on each forearm. One forearm wound was treated with topical regular insulin (Iletin-II) and the other with normal saline four times a day until healed. Treatment was double-blind and forearms were assigned randomly. The wounds treated with insulin healed 2.4 +/- 0.8 days faster than the wounds treated with saline (P < 0.001 by paired t-test). Zinc is used to crystallise insulin. When wounds are treated with insulin, they are therefore also being treated with zinc. If insulin accelerates wound healing, it is not clear if the increase in the rate of healing would be due to insulin (a known growth factor), the zinc it contains, or a combination of the two. The second study used a randomised, double-blind, placebo-controlled design to compare the efficacy of insulin with that of a solution containing the same amount of zinc in accelerating the healing of standardised wounds in rats and humans. Although these pilot investigations did not have the power to define the relative roles of insulin and zinc with accuracy, the results suggest that zinc does play a role in the wound healing process. It is concluded that topical insulin accelerates wound healing in humans. More importantly, however, this study describes a method of creating uniform wounds in humans acceptable to an institutional review board, thus solving one of the major impediments to the scientific evaluation of human wound healing.     

皮肤老化及细胞生长因子在美肤方面的应用

皮肤老化受到多种因素的影响,是当前医学美容研究的热点之一。细胞生长因子是一类刺激细胞分裂的生物活性多肽,在创伤修复过程中具有多种重要作用：促进细胞增殖、促进创面的血管化、调节细胞外基质的产生和降解、诱导邻近细胞合成细胞因子等。本文介绍了皮肤衰老的机理、细胞生长因子的特点及其在美容方面的应用。     

PERSPECTIVE ARTICLE: Growth factors and cytokines in wound healing

Wound healing is an evolutionarily conserved, complex, multicellular process that, in skin, aims at barrier restoration. This process involves the coordinated efforts of several cell types including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. The migration, infiltration, proliferation, and differentiation of these cells will culminate in an inflammatory response, the formation of new tissue and ultimately wound closure. This complex process is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines and chemokines. Of particular importance is the epidermal growth factor (EGF) family, transforming growth factor beta (TGF‐β) family, fibroblast growth factor (FGF) family, vascular endothelial growth factor (VEGF), granulocyte macrophage colony stimulating factor (GM‐CSF), platelet‐derived growth factor (PDGF), connective tissue growth factor (CTGF), interleukin (IL) family, and tumor nerosis factor‐α family. Currently, patients are treated by three growth factors: PDGF‐BB, bFGF, and GM‐CSF. Only PDGF‐BB has successfully completed randomized clinical trials in the Unites States. With gene therapy now in clinical trial and the discovery of biodegradable polymers, fibrin mesh, and human collagen serving as potential delivery systems other growth factors may soon be available to patients. This review will focus on the specific roles of these growth factors and cytokines during the wound healing process.     

Diabetes impairs adipose tissue–derived stem cell function and efficiency in promoting wound healing

Adipose tissue–derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue–derived cells from streptozotocin‐induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker‐positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)‐A, and insulin‐like growth factor‐1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers.     

Tissue expression of transforming growth factor-β1 and transforming growth factor-α during wound healing in human skin explants

In the dynamic and complex process of wound healing, locally produced growth factors are important mediators, although their actual roles have not been fully established. In the present study, the presence of transforming growth factor-beta1 and -alpha during the re-epithelialization of full-thickness wounds was investigated in an in vitro model of wound healing in human skin. The amounts of transforming growth factor-beta1 and -alpha secreted from the wound area were measured with enzyme immunoassays, and immunohistochemistry was used to study the localization of these two growth factors in the healing wound. The wounds were followed until they were completely re-epithelialized. The results showed a continuous increase in secreted transforming growth factor-beta1 throughout the re-epithelialization phase of healing followed by a decrease after its completion. The keratinocytes migrating out from the wound edges showed intense staining for transforming growth factor-beta1 which declined to the level of the surrounding epidermis after the wound was covered by a new epidermis. After the skin was wounded, a decrease both in secreted transforming growth factor-alpha and in immunostaining for this growth factor was apparent. Even though a minor increase in the immunoreactivity for transforming growth factor-alpha occurred after the completion of re-epithelialization, no increase in secreted transforming growth factor-alpha could be detected by enzyme immunoassay. These data suggest that keratinocytes modulate their expression of transforming growth factor-beta1 and -alpha during the wound healing process in human skin and that these changes may be controlled in part by autocrine pathways.     

In vitro comparative evaluation of recombinant growth factors for tissue engineering of bladder in patients with neurogenic bladder

Background

To compare the effects of various recombinant growth factors on bladder regeneration and angiogenesis for tissue engineering of bladder in patients with neurogenic bladder through in vitro cellular biological methods.

Materials and methods

Human bladder smooth muscle cells (HBSMCs) and human bladder urothelial cells (HBUCs) were cultured from patients with neurogenic bladder and used for comparative evaluations of various growth factors. Human umbilical vein endothelial cells (HUVECs) were also used. Eight potential growth factors, platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGF-β1), were selected and their effects on the proliferation, migration, and wound healing of HBSMCs, HBUCs, and HUVECs were compared.

Results

PDGF-BB, PDGF-CC, bFGF, VEGF, IGF-1, or HGF enhanced the proliferation, migration, and wound healing of HBSMCs, whereas TGF-β1 inhibited their proliferation. Proliferation, migration, and wound healing of HBUCs and HUVECs were enhanced by bFGF, VEGF, EGF, IGF-1, or HGF, whereas inhibited by TGF-β1. PDGF-BB failed to enhance cell activity of HUVECs, whereas PDGF-CC could enhance their migration and wound healing. PDGF-BB, EGF, and VEGF were the most potent factors for stimulating the activities of HBSMCs, HBUCs, and HUVECs, respectively.

Conclusions

Our findings suggest the potential use of a combination of PDGF-BB, EGF, and VEGF for bladder regeneration and angiogenesis. The synergetic effects of the three growth factors on cell activities in a three-dimensional scaffold and an animal model with neurogenic bladder need to be further evaluated.     

Bone marrow mesenchymal stem cells undergo nemosis and induce keratinocyte wound healing utilizing the HGF/c-Met/PI3K pathway

We previously showed cell–cell contacts of human dermal fibroblasts to induce expression of the hepatocyte growth factor/scatter factor (HGF) in a process designated as nemosis. Now we report on nemosis initiation in bone marrow mesenchymal stem cells (BMSCs). Because BMSCs are being used increasingly in cell transplantation therapy we aimed to demonstrate a functional effect and benefit of BMSC nemosis for wound healing. Nemotic and monolayer cells were used to stimulate HaCaT keratinocyte migration in a scratch-wound healing assay. Both indicators of nemosis, HGF production and cyclooxygenase-2 expression, were induced in BMSC spheroids. When compared with a similar amount of cells as monolayer, nemotic cells induced keratinocyte in vitro scratch-wound healing in a concentration-dependent manner. The HGF receptor, c-Met, was rapidly phosphorylated in the nemosis-stimulated keratinocytes. Nemosis-induced in vitro scratch-wound healing was inhibited by an HGF-neutralizing antibody as well as the small molecule c-Met inhibitor, SU11274. HGF-induced in vitro scratch-wound healing was inhibited by PI3K inhibitors, wortmannin and LY294002, while LY303511, an inactive structural analogue of LY294002, had no effect. Inhibitors of the mitogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126), and p38 (SB203580) attenuated HGF-induced keratinocyte in vitro scratch-wound healing. We conclude that nemosis of BMSCs can induce keratinocyte in vitro scratch-wound healing, and that in this effect signaling via HGF/c-Met is involved.     

Role of neuropeptides,neurotrophins, and neurohormones in skin wound healing

Due to the close interactions between the skin and peripheral nervous system, there is increasing evidence that the cutaneous innervation is an important modulator of the normal wound healing process. The communication between sensory neurons and skin cells involves a variety of molecules (neuropeptides, neurohormones, and neurotrophins) and their specific receptors expressed by both neuronal and nonneuronal skin cells. It is well established that neurotransmitters and nerve growth factors released in skin have immunoregulatory roles and can exert mitogenic actions; they could also influence the functions of the different skin cell types during the wound healing process.     

Stroma-epithelium crosstalk in prostate cancer

The critical role played by stroma-epithelium crosstalk in carcinogenesis and progression of prostate cancer has been increasingly recognized. These interactions are mediated by a variety of paracrine factors secreted by cancer cells and/or stromal cells. In human prostate cancer, reactive stroma is characterized by an increase in myofibroblasts and a corresponding amplification of extracellular matrix production and angiogenesis. Permanent genetic mutations have been reported in stromal cells as well as in tumour cells. Transforming growth factor-J3, vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor signalling pathways are involved in the process of angiogenesis, whereas hepatocyte growth factor, insulin-like growth factor-l, epidermal growth factor, CXC12 and Interleukin-6 play active roles in the progression, androgen-independent conversion and distal metastasis of prostate cancer. Some soluble factors have reciprocal interactions with androgens and the androgen receptor （AR）, and can even activate AR in the absence of the androgen ligand. In this article, we review the complex interactions between cancer cells and the surrounding microenvironment, and discuss the potential therapeutic targets in the stromal compartment of prostate cancer.     

生物型人工皮肤及复合bFGF对兔创面愈合的影响

目的：探讨生物型人工皮肤复合碱性成纤维生长因子对兔创面愈合的影响。方法：以动物膜组织为基材,经环氧交联固定及表面透明质酸修饰制备生物型人工皮肤,复合碱性成纤维生长因子形成生物活性人工皮肤。光镜观察生物型人工皮肤的结构,进行物理及生物性能测定水蒸汽透过量、液体吸收量、力学性能及细胞毒性。最后将两种人工皮肤植入兔全层皮肤创伤模型,观察术后4周内创面愈合情况,进行病理组织学检查评估创面的修复质量。结果：光镜下生物型人工皮肤为无细胞的胶原结构。贴附性好,无免疫排斥反应,有一定韧性,水蒸汽透过量：372±45g/（m2×24h）,液体吸收量：269±38%,无细胞毒性。复合少量碱性成纤维生长因子后,明显促进创面愈合,减少瘢痕形成。结论：生物型人工皮肤复合少量碱性成纤维生长因子使创面快速愈合,可作为烧伤科、整形外科进行皮肤重建的良好替代物。     

Proteinases, their inhibitors, and cytokine profiles in acute wound fluid

Wound healing is a complex process involving the interactions of many different cell types, matrix components and biological factors, including proteinases and cytokines. This study compared the levels of proteinases (matrix metalloproteinases and plasminogen activators), proteinase inhibitors (tissue inhibitors of metalloproteinases and plasminogen activator inhibitors), inflammatory cytokines and growth factors in acute wound fluid samples collected from the surgical drains of elective breast (n = 24) and colorectal (n = 26) patients on the first postoperative day. Gelatin zymography was used to determine matrix metalloproteinase-2 and -9 levels, quenched fluorescence substrate hydrolysis was applied for total MMP activity and enzyme-linked immunoassays were used to quantitate other factors. Colorectal wound fluid samples showed significantly (p < 0.05) greater levels of the matrix metalloproteinases (MMP-1, 2, 3, and 9), tissue inhibitor of metalloproteinases-1, urokinase plasminogen activator receptor and the inflammatory cytokines (interleukin-1beta, -6, and tumor necrosis factor-alpha); e.g., matrix metalloproteinase-3 colon; median 275 (range 11-2.530) ng/ml; breast; 530-400. However, tissue plasminogen activator and growth factor levels (epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, transforming growth factor-beta1) were significantly greater in breast samples; e.g., epidermal growth factor breast 468 (103-1, 444) pg/ml; colon 57(1-573). There was no difference in the levels of urokinase type plasminogen activator, plasminogen activator inhibitor-1 and -2, tissue inhibitor of metalloproteinases -2 or vascular endothelial growth factor. Acute wound fluid from different surgical wounds showed different profiles of proteinases, proteinase inhibitors, and cytokines. This may lead to differences in the rate of tissue remodeling and therefore healing in these two wounds in vivo.