Elsevier

Biomaterials

Volume 29, Issue 21, July 2008, Pages 3091-3104
Biomaterials

Development of biodegradable electrospun scaffolds for dermal replacement

https://doi.org/10.1016/j.biomaterials.2008.03.037Get rights and content

Abstract

Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA + 10% oligolactide; (3) PLLA + rhodamine and (4–6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.

Introduction

Our aim is to develop biodegradable electrospun dermal scaffolds to replace donor human dermis or bovine collagen for 3D skin reconstruction for future clinical use. While there have been significant advances in expanding skin cells and delivering them to patients for the treatment of extensive skin loss due to burns injury or chronic wounds [1], [2], [3], [4], [5] one major challenge which remains is the production of 3D tissue engineered skin which has both an epidermal and a dermal layer. In the treatment of extensive full thickness burns where sufficient autologous skin grafts are not immediately available for the patient the dermal and epidermal layers are often replaced in two separate operations (as described in Refs [6], [7]). The majority of dermal replacement materials currently in use are based on scaffolds of bovine collagen (e.g. Integra [7] or human donor allodermis [6]).

In the US, bovine collagen has been approved by the FDA for clinical use in tissue engineered products. For example Apligraf, which consists of bovine collagen and allogeneic keratinocytes and fibroblasts, is used for the treatment of chronic wounds [8]. Similarly Integra, which consists of bovine collagen covered by a silastic membrane to give a temporary epidermal type of barrier, is used as a dermal replacement material in the treatment of full thickness burns [7]. Bovine collagen, while extensively used and approved by the FDA, is not entirely risk free. In December 2003 there were press release reports of bovine spongiform encephalitis occurring in the US. Against this background avoiding the clinical use of bovine derived products should be considered in the future design of tissue engineered products.

Significant progress has also been made using donor skin which is decellularised and sterilised and then repopulated with the patient's own laboratory expanded keratinocytes and fibroblasts [5], [9], [12]. However, even when sourced from accredited skin banks, donor skin will always have a small but appreciable risk of viral transmission. There are also practical difficulties in sourcing donor skin and many patients and surgeons view donor skin as being acceptable only in extremely life threatening conditions. In many other conditions where it could benefit patients, e.g. contracture release and scar revision, it is not commonly used. In contrast, Integra is viewed as less of a risk, but requires a second operation to achieve epithelial cover with a thin split-thickness skin graft. A synthetic dermal matrix substitute which could also be seeded with the patients own keratinocytes and fibroblasts would decrease the risk of disease transmission for the patient and lead to a greater clinical uptake of tissue engineered skin and oral mucosa.

Of the various approaches for producing a dermal replacement, electrospun scaffolds are attractive for a number of reasons. Electrospinning can be used to produce a 3D open porous structure which approximates the structure of collagenous dermis. It is also possible to electrospin natural materials such as collagen and chitosan or synthetic materials such as poly-l-lactide (PLLA), polycaprolactone (PCL) and polyglycolic acid (PGA) [13]. Blends of both natural and synthetic materials too have been used [14]. The above materials have been approved by the FDA and used clinically for a number of years for a wide range of applications such as resorbable sutures, fracture plates and stents [15]. There is also considerable information on the degradation of PLLA and PGA which are hydrolysed into lactic and glycolic acids, respectively, and metabolised in vivo [16]. In general terms, copolymers of poly(d,l)-lactide-co-glycolide (PLGA) degrade at a faster rate than pure poly(d,l-lactide) (P(d,l)LA), but slower than pure PGA. Thus, degradation rate can be carefully controlled according to copolymer composition [13].

For the development of dermal replacement materials for clinical use in skin and oral mucosa tissue engineering, we suggest that these materials should degrade within a few months and be replaced by neo-dermis produced by the patients skin or oral mucosa cells. The degradation rate of a polymer scaffold and the manner of its degradation in vivo are the keys to achieve tissue reconstruction. As the environment encountered by a scaffold is chemically dynamic, non-equilibrium and multi-component a detailed examination of promising materials must be made. For electrospun scaffolds, fibre diameter, the interfibre space and the extent to which the scaffolds are infiltrated and vascularised influence the degradation rate in vivo. However, there is little work on the degradation rate of PLGA electrospun scaffolds in vivo. Accordingly, we report on the biodegradability of electrospun scaffolds in which we have varied the ratio of PGA to P(d,l)LA-co-PGA – referred to as PLGA polymers throughout the paper. We examined the breakdown of scaffolds under cell-free conditions in vitro and in vivo by subcutaneous implantation into rats. Data from this work were then used to select relevant scaffold materials to support both skin cell growth in vitro and the ability to synthesise production of extracellular matrix.

Section snippets

Polymers

For this study four polymers were investigated; poly(l-lactide) (PLLA) (Mn  99K) (supplied by Fluka) and three poly(d,l-lactide-co-glycolide) (PLGA) copolymers with various ratios of lactide to glycolide (85:15 (Mw 50–75K), 75:25 (Mw 66–107K) and 50:50 (Mw 40–75K)). All the PLGA copolymers were from Sigma–Aldrich. The polymers were dissolved in dichloromethane (DCM) to form solutions of suitable viscosities for electrospinning; PLLA to form an 8% (wt/wt) solution, PLGA 85:15, 75:25 and 50:50 to

Electrospinning of scaffolds

Fig. 1 shows scanning electron micrographs of scaffolds prior to implantation. Electrospun mats were 0.1–0.2 mm in thickness and all scaffolds exhibited similar surface characteristics when viewed under SEM. Polymer electrospinning produced non-woven randomly arranged fibres with an average diameter of 2.1 ± 1.2 μm for PLLA (mean ± SD), 2.9 ± 1.3 for PLLA plus lactide oligomers, 2.7 ± 0.5 μm for PLGA 85:15 scaffolds, 4.5 ± 1.4 for PLGA 75:25, and 4.3 ± 1.5 μm for PLGA 50:50 as summarised in Table 2. The pore

Discussion

The aim of this study was to develop biodegradable electrospun scaffolds as candidate materials to replace the use of human donor dermis or bovine collagen for skin reconstruction purposes. In brief, this study shows that it was possible to produce scaffolds composed of PLGA copolymers with fibre diameters of around 2–5 μm and over 95% porosity. Extensive in vitro studies showed skin cells readily attached and grew within these scaffolds, co-cultures of keratinocytes and fibroblasts were capable

Acknowledgements

We thank BBSRC for supporting this study.

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