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

https://doi.org/10.1016/S1357-2725(02)00062-6Get rights and content

Abstract

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.

Introduction

Delayed wound repair represents a significant burden for the patient, those involved with treatment and those responsible for defraying the costs of that treatment. Irrespective of the underlying disease, chronic wounds are invariably characterised by a relative inability to generate new tissue. Advances in the fields of recombinant protein technology and tissue engineering have lead to the development of a variety of topical growth factor preparations [1] and skin substitutes [2], [3]; designed to stimulate tissue formation and replace lost tissue, respectively. While some of these high technology approaches have shown a great deal of promise in recent clinical trials, cost effectiveness and efficacy issues, relative to standard clinical practice, may act to limit their uptake. The development of interactive devices, that promote healing by correcting imbalances of the wound environment, may represent a more appropriate and cost effective alternative to these high technology therapies.

While our understanding of chronic wound pathophysiology is far from complete, there is evidence to suggest that a number of factors, intrinsic to such wounds, act to impair tissue synthesis, assembly and maintenance. The lack of new tissue deposition in non-healing wounds is thought to be the consequence of both impaired tissue synthesis and elevated tissue destruction. Reduced tissue generation may be the result of a variety of derangements including phenotypic abnormalities in synthetic cells [4], [5], compromised intracellular signalling [6], [7], [8] and reduced cellular viability; the latter being explained, in part, by the damaging effects of free radicals and metal ions such as iron [9], [10], [11], [12], [13], [14]. The destructive action of elevated levels of a variety of proteinases is thought to be a key factor in the impaired healing of a variety of chronic wounds [15], [16]. Excessive proteinase activity is thought to deprive chronic wounds of initial matrix, which acts both as a conduit for cellular migration and a template for matrix deposition and growth factor sequestration, and growth and regulatory factors, which are centrally involved with the orchestration of the new tissue formation process [17], [18], [19], [20], [21], [22]. High protease activity within chronic wounds may also damage cell surface proteins such a growth factor receptors and integrins [23], [24], [25]. Proteinase-associated damage of this nature would be expected to impact on the activities of all cells involved in the tissue repair process.

Taking into consideration these known defects of the chronic wound environment, the ideal performance characteristics of materials designed to promote tissue generation within chronic wounds should include: (i) the ability to attract cells capable of synthesizing new tissue to the wound site, (ii) the ability to promote cell proliferation, (iii) the ability to offer a biocompatible and transient (bioresorbable) template for cellular migration and matrix deposition, (iv) the ability to limit excessive degradative proteinase activity, (v) the ability to adsorb and neutralise free radicals and/or excess metal ions. Oxidised regenerated cellulose (ORC)/collagen was developed with these “ideal performance properties” in mind. ORC/collagen is a bioresorbable amorphous open-pored matrix constructed of ORC (45%) and bovine collagen (55%). The ORC component, which is made by controlled milling of the haemostat SURGICEL™ was selected on the basis that it is both bioresorbable and biocompatible, with a 40 years history of safe clinical use and has a chemical profile similar to other macromolecules proven to exhibit good free radical absorption [26], [27]. The collagen component was chosen as it is predictably bioresorbable, has an established track record as a biomedical device, particularly in the field of tissue repair [28], and provides a robust protein “sink” for protease binding [29]. In a previous study [30], we have shown that this proprietary material can both bind and inactivate proteases, and adsorb free radicals/metal ions in in vitro model systems. We have also shown that it can effectively bind and protect a model growth factor (PDGF) in such a way that, in the chronic wound environment the stability of this growth factor may be improved and its activity in vivo enhanced.

The aim of the present study was to evaluate the ability of ORC/collagen to promote wound healing under impaired conditions. As fibroblasts are known to play a key role in the repair and remodelling of tissues in dermal wounds, the chemokinetic and proliferative effects of ORC/collagen on this cell lineage were investigated by in vitro assays. The capacity of ORC/collagen to promote granulation tissue formation and wound closure in the C57BL/6 KsJ (db/db) mouse, a widely used model of impaired wound healing was also investigated. While it is appreciated that no animal model, yet considered, fully encompasses all of the pathophysiological derangements associated with any form of chronic wound in man, all mammals share the basic biological processes of wound repair. That being the case, agents or devices found to augment defective repair in non-human models might similarly promote wound repair in the clinical setting.

Section snippets

Preparation of ORC/collagen sponges

Purified collagen was prepared by extensively washing the lower split (corium) of bovine hide. The dermis was cut into 10 cm squares and washed in a 0.4% (v/v) sodium hydroxide solution for 10 days, to remove residual hair and non-collagenous components. After washing, these squares were chopped and homogenised in 0.05 M acetic acid to give a final concentration of 4% (w/v) solids. ORC powder, produced by milling SURGICEL™ cloth (Ethicon Inc., NJ, USA), was added to the collagen suspension at a

The chemokinetic effect of ORC/collagen

The chemokinetic effect of ORC/collagen on human dermal fibroblasts is shown in Fig. 1. DMEM (negative control) elicited minimal cell migration, whilst in general, fibronectin (10 μg/ml) produced the maximum cell migration seen in this assay. Exposure to an ORC/collagen extract provoked extensive cell migration when applied in its undiluted “neat” form (1 mg/ml), with decreased cell migration as the dilution was increased. In this system, ORC/collagen was found to be chemokinetic for human dermal

Discussion

In a previous study [30], we showed that a biomaterial made from a combination of ORC and collagen could absorb a range of factors which are known to be present in chronic wounds and which could perturb the healing process. These included proteases, free radicals and iron ions. In the same study, we demonstrated that ORC/collagen could bind and protect PDGF whilst maintaining its biological activity, a function that may be beneficial in the chronic wound environment.

In this study, we have shown

References (67)

  • A Sattar et al.

    Application of angiogenic oligosaccharides of hyaluronan increases blood vessel numbers in rat skin

    J. Invest. Dermatol.

    (1994)
  • S.E Stringer et al.

    Heparan sulphate

    Int. J. Biochem. Cell Biol.

    (1997)
  • D.L Doxey et al.

    Platelet-derived growth factor levels in wounds of diabetic rats

    Life Sci.

    (1995)
  • M.S Bitar et al.

    Transforming growth factor-β and insulin-like growth factor-I in relation to diabetes-induced impairment of wound healing

    J. Surg. Res.

    (1996)
  • H.D Beer et al.

    Reduced expression of PDGF and PDGF receptors during impaired wound healing

    J. Invest. Dermatol.

    (1997)
  • D.L Brown et al.

    Apoptosis down-regulates inflammation under the advancing epithelial wound edge: delayed patterns in diabetes and improvement with topical growth factors

    Surgery

    (1997)
  • P.J Hennessey et al.

    EGF increases short-term type I collagen accumulation during wound healing in diabetic rats

    J. Pediatr. Surg.

    (1990)
  • T.J Fahey et al.

    Diabetes impairs the late inflammatory response to wound healing

    J. Surg. Res.

    (1991)
  • S Werner et al.

    Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse

    J. Invest. Dermatol.

    (1994)
  • R.M Kuntz et al.

    Neutrophil motility in extracellular matrix gels: mesh size and adhesion affect speed of migration

    Biophys. J.

    (1997)
  • F Grinnell et al.

    Degradation of fibronectin and vitronectin in chronic wound fluid: analysis by cell blotting, immunoblotting, and cell adhesion assays

    J. Invest. Dermatol.

    (1992)
  • E.C Bullen et al.

    Tissue inhibitor of metalloproteinases-1 is decreased and activated gelatinases are increased in chronic wounds

    J. Invest. Dermatol.

    (1995)
  • M Weckroth et al.

    Matrix metallo-proteinases, gelatinase and collagenase, in chronic leg ulcers

    J. Invest. Dermatol.

    (1996)
  • B.C Nwomeh et al.

    MMP-8 is the predominant collagenase in healing wounds and non-healing ulcers

    J. Surg. Res.

    (1999)
  • G Lauer et al.

    Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds

    J. Invest. Dermatol.

    (2000)
  • J. Hart, Growth factors, in: M. Miller, D. Glover (Eds.), Wound Management Theory and Practice, N.T. Books, London,...
  • M.V Sefton et al.

    Tissue engineering

    A. J. Cutan. Med. Surg.

    (1998)
  • D.L Stocum

    Regenerative biology and engineering: strategies for tissue restoration

    Wound Rep. Reg.

    (1998)
  • P Schmid et al.

    TGF-β and TGF-β type II receptor in human epidermis: differential expression in acute and chronic skin wounds

    J. Pathol.

    (1993)
  • G.F Pierce et al.

    Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic non-healing wounds

    J. Clin. Invest.

    (1995)
  • D.R Yager 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. Reg.

    (1997)
  • N Borregaard et al.

    Prevention of tissue damage: inhibition of myeloperoxidase mediated inactivation of α 1-proteianse inhibitor by N-acetyl cysteine, glutathione and methionine

    Agents Actions

    (1987)
  • M. Symons, J. Gutteridge, Free radicals and Iron: Chemistry, Biology and Medicine, Oxford Science Publications, Oxford,...
  • Cited by (100)

    • Control of Staphylococcal-mediated endogenous protease activity alters wound closure time in a complex wound model

      2022, Journal of Dermatological Science
      Citation Excerpt :

      They are composed of 55% collagen and 45% ORC, with Prisma® dressings also containing 1% silver-ORC. Studies have shown that Promogran® can bind and inactivate elastase, plasmin and metalloproteinase activity in chronic wound exudates of diabetic patients in vitro [14], and accelerate wound repair in a diabetic mouse model [15]. Randomised controlled trials, studying diabetic foot ulcers, pressure ulcers and venous leg ulcers, have all reported a significant increase in the rate of wound healing after treatment with ORC/collagen dressings (summarised in [16]).

    • Current Trends in the Development of Wound Dressings, Biomaterials and Devices

      2022, Encyclopedia of Materials: Plastics and Polymers
    View all citing articles on Scopus
    View full text