The role of oxidised regenerated cellulose/collagen in wound repair: effects in vitro on fibroblast biology and in vivo in a model of compromised healing

Irrespective of underlying chronic wound pathology, delayed wound healing is normally characterised by impaired new tissue formation at the site of injury. It is thought that this impairment reflects both a reduced capacity to synthesize new tissue and the antagonistic activities of high levels of proteinases within the chronic wound environment. Historically, wound dressings have largely been passive devices that offer the wound interim barrier function and establish a moist healing environment. A new generation of devices, designed to interact with the wound and promote new tissue formation, is currently being developed and tested. This study considers one such device, oxidised regenerated cellulose (ORC) /collagen, in terms of its ability to promote fibroblast migration and proliferation in vitro and to accelerate wound repair in the diabetic mouse, a model of delayed wound healing. ORC/collagen was found to promote both human dermal fibroblasts proliferation and cell migration. In vivo studies considered the closure and histological characteristics of diabetic wounds treated with ORC/collagen compared to those of wounds given standard treatment on both diabetic and non-diabetic mice. ORC/collagen was found to significantly accelerate diabetic wound closure and result in a measurable improvement in the histological appearance of wound tissues. As the diabetic mouse is a recognised model of impaired healing, which may share some characteristics of human chronic wounds, the results of this in vivo study, taken together with those relating the positive effects of ORC/collagen in vitro, may predict the beneficial use of this device in the clinical setting.     

Immunohistochemical localization of growth factors in fetal wound healing

Fetal wound healing occurs rapidly, in a regenerative fashion, and without scar formation, by contrast with adult wound healing, where tissue repair results in scar formation which limits tissue function and growth. The extracellular matrix deposited in fetal wounds contains essentially the same structural components as that in the adult wound but there are distinct differences in the spatial and temporal distribution of these components. In particular the organization of collagen in the healed fetal wound is indistinguishable from the normal surrounding tissue. Rapidity of healing, lack of an inflammatory response, and an absence of neovascularization also distinguish fetal from adult wound healing. The mechanisms controlling these differing processes are undefined but growth factors may play a critical role. The distribution of growth factors in healing fetal wounds is unknown. We have studied, by immunohistochemistry, the localization of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and basic fibroblast growth factor (bFGF), in fetal, neonatal, and adult mouse lip wounds. TGF beta and bFGF were present in neonatal and adult wounds, but were not detected in the fetal wounds, while PDGF was present in fetal, neonatal, and adult wounds. This pattern correlates with the known effects in vitro of these factors, the absence of an inflammatory response and neovascularization in the fetal wound, and the patterns of collagen deposition in both fetal and adult wounds. The results suggest that it may be possible to manipulate the adult wound to produce more fetal-like, scarless, wound healing.     

Proteome analysis reveals antiangiogenic environments in chronic wounds of diabetes mellitus type 2 patients

In contrast to normal healing wounds, chronic wounds commonly show disturbances in proteins regulating wound healing processes, particularly those involved in cell proliferation and protein degradation. Multidimensional protein identification technology MS/MS was conducted to investigate and compare the protein composition of chronic diabetic foot exudates to exudates from split‐skin donor sites of burn victims otherwise healthy. Spectral counting revealed 188 proteins differentially expressed (more than twofold and p‐value <0.05) in chronic wounds. Most were involved in biological processes including inflammation, angiogenesis, and cell mortality. Increased expression of the inflammatory response stimulating S100 proteins, predominantly S100A8 and S100A9 (almost tenfold), was identified. Matrix metalloproteinases (MMPs) MMP1, MMP2, and MMP8 were identified to be elevated in chronic wounds with significant impact on collagen degradation and tissue destruction. Further, proteins with antiangiogenic properties were found at higher expression levels in chronic wounds. Reduced angiogenesis leads to drastic shortage in nutrition supply and causes increased cell death, demonstrated by Annexin A5 exclusively found in chronic wound exudates. However, excessive nucleic and cytosolic material infers cell death occurring not only by apoptosis but also by necrosis. In conclusion, mass spectrometric investigation of exudates from chronic wounds demonstrated dramatic impairment in wound repair with excessive inflammation, antiangiogenic environment, and accelerated cell death.     

Role of platelet-derived growth factor in wound healing

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and fibroblasts in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as 20-200 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and fibroblasts and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other growth factors, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide growth factors may be the critical regulators of extracellular matrix deposition within healing wounds.     

Wound healing enhancement in leg ulcers: A case report

Wound healing in the skin is a complex biological process in which numerous types of cells, cytokines, growth factors, proteases and extracellular matrix components act in concert to restore the integrity of injured tissue. Cultivated allogenic human keratinocytes have been used for the treatment of various skin defects like burnwounds, surgical wounds, in exfoliative skin diseases and chronic wounds. A new method for wound healing enhancement in leg ulcers using cultured allogenic keratinocytes suspended in fibrin glue and used in spray technique is introduced. Allogenic keratinocytes are supposed to enhance granulation tissue production and to stimulate reepithelisation due to their release of growth factors and thus are able to recreate an active wound. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Novel Model of Chronic Wounds: Importance of Redox Imbalance and Biofilm-Forming Bacteria for Establishment of Chronicity

Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone [1]. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.     

Calpain activity is essential in skin wound healing and contributes to scar formation

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFβ-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.     

The effect of nerve growth factor on the early responses during the process of wound healing

In this study, we investigated the role of nerve growth factor (NGF)-incorporated collagen on wound healing in rats. Full-thickness excision wounds were made on the back of female rats weighing about 150-160 g. Topical application of NGF-incorporated collagen, at a concentration of 1 microg/1.2 mg collagen/cm(2), once a day, for 10 days resulted in complete healing of wounds on the 15th day. The concentrations of collagen, hexosamine and uronic acid in the granulation tissue were determined. The NGF-incorporated collagen-treated rats required shorter duration for the healing with an increased rate of wound contraction. Histological and electron microscopical evaluations were also performed, which reveal the activation of fibroblasts and endoplasmic reticulum and therefore increased level of collagen synthesis due to NGF application. These results clearly indicate that the topical application of NGF-incorporated collagen enhanced the rate of healing of excision wounds.     

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ?mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.     

Dysregulation of monocyte/macrophage phenotype in wounds of diabetic mice

The hypothesis of this study was that cells of the monocyte/macrophage lineage (Mo/Mp) exhibit an impaired transition from pro-inflammatory to pro-healing phenotypes in wounds of diabetic mice, which contributes to deficient healing. Mo/Mp isolated from excisional wounds in non-diabetic db/+ mice exhibited a pro-inflammatory phenotype on day 5 post-injury, with high level expression of the pro-inflammatory molecules interleukin-1β, matrix metalloprotease-9 and inducible nitric oxide synthase. Wound Mo/Mp exhibited a less inflammatory phenotype on day 10 post-injury, with decreased expression of the pro-inflammatory molecules and increased expression of the alternative activation markers CD206 and CD36. In contrast, in db/db mice, the pro-inflammatory phenotype persisted through day 10 post-injury and was associated with reduced expression of insulin-like growth factor-1, transforming growth factor-β1 and vascular endothelial growth factor. Reduced levels of these growth factors in wounds of db/db mice may have contributed to impaired wound closure, reduced granulation tissue formation, angiogenesis and collagen deposition. The persistent pro-inflammatory wound Mo/Mp phenotype in db/db mice may have resulted from elevated levels of pro-inflammatory interleukin-1β and interferon-γ and reduced levels of anti-inflammatory interleukin-10 in the wound environment. Our findings are consistent with the hypothesis that dysregulation of Mo/Mp phenotypes contributes to impaired healing of diabetic wounds.     

Modelling the interaction of keratinocytes and fibroblasts during normal and abnormal wound healing processes

The crosstalk between fibroblasts and keratinocytes is a vital component of the wound healing process, and involves the activity of a number of growth factors and cytokines. In this work, we develop a mathematical model of this crosstalk in order to elucidate the effects of these interactions on the regeneration of collagen in a wound that heals by second intention. We consider the role of four components that strongly affect this process: transforming growth factor-β, platelet-derived growth factor, interleukin-1 and keratinocyte growth factor. The impact of this network of interactions on the degradation of an initial fibrin clot, as well as its subsequent replacement by a matrix that is mainly composed of collagen, is described through an eight-component system of nonlinear partial differential equations. Numerical results, obtained in a two-dimensional domain, highlight key aspects of this multifarious process, such as re-epithelialization. The model is shown to reproduce many of the important features of normal wound healing. In addition, we use the model to simulate the treatment of two pathological cases: chronic hypoxia, which can lead to chronic wounds; and prolonged inflammation, which has been shown to lead to hypertrophic scarring. We find that our model predictions are qualitatively in agreement with previously reported observations and provide an alternative pathway for gaining insight into this complex biological process.     

Mechanical unloading impairs keratinocyte migration and angiogenesis during cutaneous wound healing.

Although initially thought to improve an individual's ability to heal, mechanical unloading promoted by extended periods of bed rest has emerged as a contributing factor to delayed or aberrant tissue repair. Using a rat hindlimb unloading (HLU) model of hypogravity, we mimicked some aspects of physical inactivity by removing weight-bearing loads from the hindlimbs and producing a systemic cephalic fluid shift. This model simulates bed rest in that the animal undergoes physiological adaptations, resulting in a reduction in exercise capability, increased frequency of orthostatic intolerance, and a reduction in plasma volume. To investigate whether changes associated with prior prolonged bed rest correlate with impaired cutaneous wound healing, we examined wound closure, angiogenesis, and collagen content in day 2 to day 21 wounds from rats exposed to HLU 2 wk before excisional wounding. Wound closure was delayed in day 2 wounds from HLU rats compared with ambulatory controls. Although the levels of proangiogenic growth factors, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF) were similar between the two groups, wound vascularity was significantly reduced in day 7 wounds from HLU animals. To further examine this disparity, total collagen content was assessed but found to be similar between the two groups. Taken together, these results suggest that keratinocyte and endothelial cell function may be impaired during the wound healing process under periods of prolonged inactivity or bed rest.     

Roles of lactoferrin on skin wound healing

Skin wound healing is a complex biological process that requires the regulation of different cell types, including immune cells, keratinocytes, fibroblasts, and endothelial cells. It consists of 5 stages: hemostasis, inflammation, granulation tissue formation, re-epithelialization, and wound remodeling. While inflammation is essential for successful wound healing, prolonged or excess inflammation can result in nonhealing chronic wounds. Lactoferrin, an iron-binding glycoprotein secreted from glandular epithelial cells into body fluids, promotes skin wound healing by enhancing the initial inflammatory phase. Lactoferrin also exhibits anti-inflammatory activity that neutralizes overabundant immune response. Accumulating evidence suggests that lactoferrin directly promotes both the formation of granulation tissue and re-epithelialization. Lactoferrin stimulates the proliferation and migration of fibroblasts and keratinocytes and enhances the synthesis of extracellular matrix components, such as collagen and hyaluronan. In an in vitro model of wound contraction, lactoferrin promoted fibroblast-mediated collagen gel contraction. These observations indicate that lactoferrin supports multiple biological processes involved in wound healing.     

Non-adenine based purines accelerate wound healing

Wound healing is a complex sequence of cellular and molecular processes that involves multiple cell types and biochemical mediators. Several growth factors have been identified that regulate tissue repair, including the neurotrophin nerve growth factor (NGF). As non-adenine based purines (NABPs) are known to promote cell proliferation and the release of growth factors, we investigated whether NABPs had an effect on wound healing. Full-thickness, excisional wound healing in healthy BALB/c mice was significantly accelerated by daily topical application of NABPs such as guanosine (50% closure by days 2.5–2.8). Co-treatment of wounds with guanosine plus anti-NGF reversed the guanosine-promoted acceleration of wound healing, indicating that this effect of guanosine is mediated, at least in part, by NGF. Selective inhibitors of the NGF-inducible serine/threonine protein kinase (protein kinase N), such as 6-methylmercaptopurine riboside abolished the acceleration of wound healing caused by guanosine, confirming that activation of this enzyme is required for this effect of guanosine. Treatment of genetically diabetic BKS.Cg-m+/+lepr db mice, which display impaired wound healing, with guanosine led to accelerated healing of skin wounds (25% closure by days 2.8–3.0). These results provide further confirmation that the NABP-mediated acceleration of cutaneous wound healing is mediated via an NGF-dependent mechanism. Thus, NABPs may offer an alternative and viable approach for the treatment of wounds in a clinical setting.     

In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen deposition, and neovascularization.

Fetal tissue repair occurs without acute inflammation, prominent fibroplasia, or marked neovascularization. The fetal wound extracellular matrix is rich in hyaluronic acid (HA), while collagen is deposited in an organized normal dermal pattern. In various biologic systems, including regeneration and development, the controlled accumulation and subsequent degradation of hyaluronic acid is associated with distinct cellular and matrix events. Therefore, it is hypothesized that the abundance of hyaluronic acid in fetal wounds may influence cellular and/or matrix events such that tissue repair is highly organized and adult-like scarring does not occur. To test this hypothesis, the hyaluronic acid content of fetal rabbit wounds was reduced by specific degradation with Streptomyces hyaluronidase. Control wounds were treated with either enzyme buffer (n = 12) or denatured enzyme solution (n = 8) and exhibited a normal fetal healing response with scattered peripheral fibroblasts, a matrix of hyaluronic acid, and no infiltrating collagen. In marked contrast, the hyaluronidase-treated wounds (n = 14) demonstrated increased fibroblast infiltration, collagen deposition, and capillary formation. A significant reduction in the hyaluronic acid content of the hyaluronidase-treated wounds was confirmed biochemically. Since the degradation of hyaluronic acid resulted in an altered healing response, this study demonstrates that hyaluronic acid affects the cellular and matrix events in fetal healing and may be partially responsible for the unique qualities of this regenerative repair process.     

Acute ethanol exposure impairs angiogenesis and the proliferative phase of wound healing

Acute ethanol exposure represents an increased risk factor for morbidity and mortality associated with surgical or traumatic injury. Despite clinical observations suggesting that ethanol exposure before injury alters tissue repair processes, little direct evidence about the mechanism by which ethanol affects the wound healing process is available. In this study, excisional wounds from female BALB/c mice with or without circulating ethanol levels of 100 mg/dl were used to assess wound closure, angiogenesis, and collagen content. Ethanol exposure resulted in a significant but transient delay in wound closure at day 2 postwounding (28 +/- 4% vs. 17 +/- 1%). In addition, total collagen content was significantly reduced by up to 37% in wounds from ethanol-treated mice compared with controls. The most significant effect of ethanol exposure on wounds was on vascularity because angiogenesis was reduced by up to 61% in wounds from ethanol-treated mice. The reduction in vessel density occurred despite near-normal levels of proangiogenic factors VEGF and FGF-2, suggesting a direct effect of ethanol exposure on endothelial cell function. Further evidence for a direct effect was observed in an in vitro angiogenesis assay because the exposure of endothelial cells to ethanol reduced angiogenic responsiveness to just 8.33% of control in a cord-forming assay. These studies provide novel information regarding the effect of a single dose of ethanol on multiple parameters of the wound healing process in vivo and suggest a potential mechanism by which ethanol impairs healing after traumatic injury.     

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100?mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.     

Dermal excisional wound healing in pigs following treatment with topically applied pure oxygen

Hypoxia, caused by disrupted vasculature and peripheral vasculopathies, is a key factor that limits dermal wound healing. Factors that can increase oxygen delivery to the regional tissue, such as supplemental oxygen, warmth, and sympathetic blockade, can accelerate healing. Clinical experience with adjunctive hyperbaric oxygen therapy (HBOT) in the treatment of chronic wounds have shown that wound hyperoxia may increase granulation tissue formation and accelerate wound contraction and secondary closure. However, HBOT is not applicable to all wound patients and may pose the risk of oxygen toxicity. Thus, the efficacy of topical oxygen treatment in an experimental setting using the pre-clinical model involving excisional dermal wound in pigs was assessed. Exposure of open dermal wounds to topical oxygen treatment increased tissue pO₂ of superficial wound tissue. Repeated treatment accelerated wound closure. Histological studies revealed that the wounds benefited from the treatment. The oxygen treated wounds showed signs of improved angiogenesis and tissue oxygenation. Topically applied pure oxygen has the potential of benefiting some wound types. Further studies testing the potential of topical oxygen in pre-clinical and clinical settings are warranted.     

Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice

Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM.     

ECM degrading proteases and tissue remodelling in the mammary gland

Matrix degradation and tissue remodelling directed by matrix-degrading proteases are activated in physiological situations such as wound healing and involution of the prostate, ovaries and uterus. Recently, other activities, in addition to the cleavage of matrix proteins, have been attributed to matrix proteases including the release of growth factors from the extracellular matrix and roles in the maturation of adipocytes. This review describes extracellular proteases, including MMPs, plasminogen and cathepsins involved in the tissue remodelling processes that occur in the breast during pubertal mammary development and the mammary cycle of pregnancy, lactation and weaning. It particularly focuses on development and weaning, termed mammary gland involution, when the majority of remodelling occurs. It also brings together recent findings on the exciting new functions of matrix-degrading proteases.