Elsevier

Biomaterials

Volume 32, Issue 21, July 2011, Pages 4782-4792
Biomaterials

A denatured collagen microfiber scaffold seeded with human fibroblasts and keratinocytes for skin grafting

https://doi.org/10.1016/j.biomaterials.2011.03.023Get rights and content

Abstract

Biomaterial scaffolds are categorized into artificial or natural polymers, or combinations of the two. Artificial polymers often undergo serum protein adsorption, elicit foreign body and encapsulation immune responses post-implantation. Large pore bovine electrospun collagen I was therefore screened as a candidate for human keratinocyte and fibroblast cell scaffolds. Human HaCaT keratinocyte and dermal fibroblasts were seeded on electrospun denatured collagen I microfiber (DCM) scaffolds and after 72 h Livedead® assays performed to determine adhesive cell, survival and scaffold penetration. Both keratinocytes and fibroblasts attached to and survived on DCM scaffolds, however only fibroblasts migrated over and into this biomaterial. HaCaT keratinocytes remained largely stationary on the scaffold surface in discrete islands of monolayered cells. For this reason, normal human epidermal keratinocyte (NHEK) scaffold interactions were assessed using scanning and transmission electron microscopy (EM) that demonstrated DCM scaffolds comprised networks of interlocking and protruding collagen fibers with a mean diameter of 2–5 μm, with a mean inter-fiber pore size of 6.7 μm (range 3–10 μm) and scaffold thickness 50–70 μm. After 72 h the keratinocytes and fibroblasts on DCM scaffolds had attached, flattened and spread over the entire scaffold with assembly of lamellapodia and focal adhesion (FA)-like junctions. Using transmission EM, NHEKs and HaCaT keratinocytes assembled desmosomes, lamellapodia and FA junctions, however, neither hemidesmosomes nor basal lamina were present. In long term (21 day) co-culture fibroblasts migrated throughout the scaffold and primary keratinocytes (and to a lesser extend HaCaTs) stratified on the scaffold surface forming a human skin equivalent (HSE). In vivo testing of these HSEs on immunocompetent (BalbC) and immunodeficient (SCID) excisionally wounded model mice demonstrated scaffold wound biocompatibility and ability to deliver human cells after scaffold biodegradation.

Introduction

Skin tissue engineering addresses the need for early, permanent coverage of extensive skin injury in burns patients with an insufficient source of autologous skin for grafting [1], [2]. Severe burn injuries require prompt wound closure but are hampered by limited patient donor site area and the high number of separate surgical operations often required to complete treatment [1], [2]. In clinical situations in which insufficient donor skin is available, bioengineered skin in the form of cultured keratinocytes or in combination with fibroblasts to form human skin equivalents (HSEs) has allowed a greater expansion of donor surface area than conventional methods [3]. Cultured skin keratinocyte sheets are typically too fragile for transfer in vitro for engraftment and are commonly supported by biomaterial scaffolds that mimic specific tissues [4], [5]. Such biomimetic scaffolds are classified into either naturally occurring [6], [7], [8] or artificial substrates [9] or combinations of the two source materials. We have previously tested artificial polymers that have many favorable properties [9], although unfortunately some polymers have generally not performed up to expectations in the clinical setting [10], [11]. Here, we examine the efficacy of a natural polymer scaffold derived from bovine collagen I. Previous biomimetic scaffolds have included freeze dried collagen sponges alone [4], sponges seeded with fibroblasts [12], collagen I scaffolds cross-linked with elastin [13], collagen II scaffolds [14], collagen and artificial polymer mixes [15], [16], [17], collagen cross-linked by carbodiimide [18] or electrospun collagen alone [19], [20], [21], electrospun collagen with epidermal keratinocytes [22] and scaffolds co-cultured with fibroblasts and keratinocytes [8], [23]. The advantages of electrospun denatured collagen microfiber (DCM) scaffolds over frozen sponges come from a more homogenous pore structure and closer biomimetic structure to naturally occurring extracellular matrix [23] with pores of 5–10 microns allowing penetration of fibroblasts into the scaffold [24].

An important function of any potential skin substitute is to support the formation of a proper epidermal barrier to limit trans-epidermal water loss, infection and reduce the chances of hypertrophic scarring by speeding wound closure and ultimately patient recovery [25]. While the need to replace the epidermal barrier is paramount, restoration of normal structure and function of dermal tissue architecture is also critical to achieve acceptable cosmetic results [25]. Currently, there are very few skin substitutes that meet all of these criteria; however, recent cultured skin substitutes comprising fibroblast and keratinocyte cells on natural scaffolds meet many graft requirements [3], [23]. The use of natural scaffolds patterned in novel ways to support reconstituted HSEs has primarily focused on reducing scar formation in animal wound models [8]. There are currently several animal wound models on which to test engraftment of human bioengineered skin composites onto immunodeficient mice to assess scarring [26]. In this study we have evaluated the use of DCM scaffold for supporting different human keratinocyte and fibroblast cell combinations for the preparation and transplantation of skin grafts from in vitro cultures. We have used in vitro cell viability assays to assess HaCaT keratinocyte and fibroblast cell attachment, survival, migration and morphology on DCM scaffolds. Due to the limitations of HaCaT cells to stratify in culture we also included primary human keratinocyte containing cultures in ultrastructural and functional HSE grafts studies. The biocompatibility of the DCM scaffold without cells was tested in excisionally wounded immunocompetent mice. In addition, the efficacy of different cell combinations of DCM composite grafts were tested in excisionally wounded immunocompromised SCID mice. Furthermore, the ability of the grafts to deliver live human cells and improve specific wound healing outcomes including reducing wound closure rates, shortening re-epithelialization times, reducing dermal foreign body and encapsulation immune responses and reducing dermal fibrosis was also assessed.

Section snippets

Preparation of biomaterial

Denatured collagen microfiber (DCM) scaffolds were manufactured as previously described using acid extraction techniques [18] and subsequently disinfected/sterilized (using ethanol and UV light sterilization), washed, air dried and vacuum stored until required. Fiber diameter and morphology of the electrospun scaffold were controlled by concentration and molecular weight of the polymer as previously described [21], [24], [27]. Fiber diameters of 3–10 μm (greater than 3–4 μm) were produced to

Livedead® cell assay

The results show that DCM scaffold is able to maintain adhesion and cell survival of both keratinocyte HaCaT cells (Fig. 1a, c, e) and fibroblasts on DCM (Fig. 1b, d, f) in a similarly effective fashion as cells cultured on conventional tissue culture plastic (TCP) (g). There were however, two main differences between keratinocyte and fibroblast migration on DCM compared to TCP. Firstly, keratinocyte cells formed discrete islands over a limited area on the DCM surface suggesting that lateral

Discussion

The material properties of electrospun denatured collagen microfiber (DCM) make this a promising candidate scaffold for skin-derived cell grafting. It comprises extracted bovine collagen and when combined with cells in a composite, avoids adverse foreign body immune responses after grafting more than many typical artificial polymers [9], [21], [34]. Bovine collagen was easy and cheap to prepare and quick to manufacture on an appropriate scale using a previously described process of acid

Conclusions

In conclusion, our data taken together show that DCM scaffold exhibits improved mechanical properties in terms of support and reducing graft shrinkage over unsupported cultured epithelial autografts (CEA) and avoids typical artificial polymer based scaffold immune responses. Furthermore, DCM cultured cell grafts do not require any donor site biopsies avoiding subsequent donor wounds as split thickness skin grafts procedures do. Further work would be beneficial to optimize keratinocyte adhesion,

Acknowledgments

This work was funded by a New Staff Start-Up Grant from The University of Queensland (JRM), by a grant-in-aid from the Health and Labor Sciences Research Grant (research into specific diseases) H17-Saisei-12 (Japan) and by support from the Brisbane Royal Children’s Hospital Foundation (Queensland Children’s Medical Research Institute, QCMRI). Author Disclosure Statement: The authors state that no completing financial interests exist.

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