Zee Upton

Increased matrix metalloproteinase‐9 (MMP‐9) activity observed in chronic wound fluid is related to the clinical severity of the ulcer

Background  The pathology of chronic wounds is often characterized by elevated levels of proinflammatory cytokines [e.g. tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β], proteases [e.g. matrix metalloproteinases (MMPs)] and neutrophil elastase. MMPs specifically have been implicated by a number of studies as the major protease family responsible for the degradation of key factors critical to the ulcer’s ability to heal.

Vitronectin: Growth Factor Complexes Hold Potential as a Wound Therapy Approach

Topical administration of growth factors has displayed some potential in wound healing, but variable efficacy, high doses, and costs have hampered their implementation. Moreover, this approach ignores the fact that wound repair is driven by interactions between multiple growth factors and extracellular matrix (ECM) proteins. We report herein that complexes comprising IGF and IGF-binding proteins bound to the ECM protein vitronectin (VN) significantly enhance cellular functions relevant to wound repair in human skin keratinocytes in two- and three-dimensional in vitro cell models and are active, even in the presence of wound fluid. Moreover, these responses require activation of both the IGF receptor and the VN-binding alpha(v) integrins. Further, we assessed the complexes as a topical agent in the treatment of deep dermal partial thickness burns in a porcine model. This pilot study revealed that the complexes may hold promise as a wound healing therapy. Critically, the significant responses observed in vitro and the encouraging preliminary data in vivo were obtained with nanogram doses of growth factors. This suggests that coupling delivery of growth factors to ECM proteins such as VN may ultimately prove to be a more effective strategy for developing a wound healing therapy.

Recent advances in dermal wound healing: biomedical device approaches

Successful repair of wounds and tissues remains a major healthcare and biomedical challenge in the 21st Century. In particular, chronic wounds often lead to loss of functional ability, increased pain and decreased quality of life, and can be a burden on carers and health-system resources. Advanced healing therapies employing biological dressings, skin substitutes, growth factor-based therapies and synthetic acellular matrices, all of which aim to correct irregular and dysfunctional cellular pathways present in chronic wounds, are becoming more popular. This review focuses on recent advances in biologically inspired devices for wound healing and includes a commentary on the challenges facing the regulatory governance of such products.

Attenuation of protease activity in chronic wound fluid with bisphosphonate-functionalised hydrogels

Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcers ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy.

Hyperbaric oxygen stimulates epidermal reconstruction in human skin equivalents

The crucial role of oxygen during the complex process of wound healing has been extensively described. In chronic or nonhealing wounds, much evidence has been reported indicating that a lack of oxygen is a major contributing factor. Although still controversial, the therapeutic application of hyperbaric oxygen (HBO) therapy can aid the healing of chronic wounds. However, how HBO affects reepithelization, involving processes such as keratinocyte proliferation and differentiation, remains unclear. We therefore used a three‐dimensional human skin–equivalent (HSE) model to investigate the effects of daily 90‐minute HBO treatments on the reconstruction of an epidermis. Epidermal markers of proliferation, differentiation, and basement membrane components associated with a developing epidermis, including p63, collagen type IV, and cytokeratins 6, 10, and 14, were evaluated. Morphometric analysis of hematoxylin and eosin‐stained cross sections revealed that HBO treatments significantly accelerated cornification of the stratum corneum compared with controls. Protein expression as determined by immunohistochemical analysis confirmed the accelerated epidermal maturation. In addition, early keratinocyte migration was enhanced by HBO. Thus, HBO treatments stimulate epidermal reconstruction in an HSE. These results further support the importance of oxygen during the process of wound healing and the potential role of HBO therapy in cutaneous wound healing.

Hyaluronic acid: evaluation as a potential delivery vehicle for vitronectin:growth factor complexes in wound healing applications.

We have previously reported that novel vitronectin:growth factor (VN:GF) complexes significantly increase re-epithelialization in a porcine deep dermal partial-thickness burn model. However, the potential exists to further enhance the healing response through combination with an appropriate delivery vehicle which facilitates sustained local release and reduced doses of VN:GF complexes. Hyaluronic acid (HA), an abundant constituent of the interstitium, is known to function as a reservoir for growth factors and other bioactive species. The physicochemical properties of HA confer it with an ability to sustain elevated pericellular concentrations of these species. This has been proposed to arise via HA prolonging interactions of the bioactive species with cell surface receptors and/or protecting them from degradation. In view of this, the potential of HA to facilitate the topical delivery of VN:GF complexes was evaluated. Two-dimensional (2D) monolayer cell cultures and 3D de-epidermised dermis (DED) human skin equivalent (HSE) models were used to test skin cell responses to HA and VN:GF complexes. Our 2D studies revealed that VN:GF complexes and HA stimulate the proliferation of human fibroblasts but not keratinocytes. Experiments in our 3D DED-HSE models showed that VN:GF complexes, both alone and in conjunction with HA, led to enhanced development of both the proliferative and differentiating layers in the DED-HSE models. However, there was no significant difference between the thicknesses of the epidermis treated with VN:GF complexes alone and VN:GF complexes together with HA. While the addition of HA did not enhance all the cellular responses to VN:GF complexes examined, it was not inhibitory, and may confer other advantages related to enhanced absorption and transport that could be beneficial in delivery of the VN:GF complexes to wounds.

3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling.

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

Preparation of cultured skin for transplantation using insulin-like growth factor I in conjunction with insulin-like growth factor binding protein 5, epidermal growth factor, and vitronectin.

Background. Cultured skin for transplantation is routinely prepared by growing patient keratinocytes in the presence of semidefined sources of growth factors including serum and feeder cells, but these materials require substantial risk remediation and can contribute to transplant rejection. Methods. We have therefore investigated the potential of a novel combination of recombinant and purified growth factors to replace serum and feeder cells in cultures of human keratinocytes suitable for clinical application. Our technique was investigated with respect to culture establishment, serial propagation, colony-forming efficiency, immunocytochemistry, epidermal reconstruction, and suitability to support transplantation by aerosolization. Results. We demonstrate that insulin-like growth factor (IGF)-I—used in conjunction with epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-5 and vitronectin—supports growth in the absence of serum. Moreover, a threefold greater number of cells are generated within 7 days compared to those grown under current best practice conditions using serum (P<0.05). The resulting test cultures are suitable for epidermal reconstruction and support the option for delivery in the form of an aerosolized cell suspension. Serial propagation, with the view to producing confluent sheets for extensive injuries, was achieved but with less consistency and this result correlated with a significant decline in colony-forming efficiency compared to controls. Conclusions. IGF-I used in conjunction with IGFBP-5, EGF, and vitronectin provides a superior alternative to serum for the rapid expansion and transplantation of cultured keratinocytes within the first week of treatment. Nevertheless, further optimization is required with respect to elimination of feeder cells and serial expansion of cultures for treatment of extensive injuries.

Vitronectin—Master controller or micromanager?

The concept that the mammalian glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin‐knockout (VN‐KO) mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN‐KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that VN occupies a role in the earliest events of thrombogenesis and tissue repair. VN is the foundation upon which the thrombus grows in an organised structure. In addition to sealing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators, and provides a provisional scaffold for the repairing tissue. In the absence of VN (e.g., VN‐KO animal), this cascade is disrupted before it begins. A wide variety of biologically active species associate with VN. Although initial studies were focused on mitogens, other classes of bioactives (e.g., glycosaminoglycans and metalloproteinases) are now also known to specifically interact with VN. Although some interactions are transient, others are long‐lived and often result in multi‐protein complexes. Multi‐protein complexes provide several advantages: prolonging molecular interactions, sustaining local concentrations, facilitating co‐stimulation of cell surface receptors and thereby enhancing cellular/biological responses. We contend that these, or equivalent, multi‐protein complexes facilitate VN polyfunctionality in vivo. It is also likely that many of the species demonstrated to associate with VN in vitro, also associate with VN in vivo in similar multi‐protein complexes. Thus, the predominant biological function of VN is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave and ‘how’ to behave (i.e., appropriately for the current circumstance).

Chimeric vitronectin:insulin-like growth factor proteins enhance cell growth and migration through co-activation of receptors.

Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies.