Viability and Function of Autologous and Allogeneic Fibroblasts Seeded in Dermal Substitutes after Implantation

doi:10.1016/j.jss.2004.11.012

Journal of Surgical Research

Volume 125, Issue 1, 1 May 2005, Pages 56-67

https://doi.org/10.1016/j.jss.2004.11.012 Get rights and content

Background

Fibroblast-seeded collagen sponges have been used for the treatment of skin defects and skin ulcers. However, the viability of the fibroblasts after implantation is still unknown. The objective of this study was to investigate the viability and distribution of autologous and allogeneic fibroblasts after implantation and to clarify which type is more effective for wound healing.

Materials and methods

Skin samples of Hartley guinea pigs were retrieved and autologous fibroblasts were isolated and cultured. Fibroblasts isolated from the skin of a Strain2 guinea pig were used as allogeneic fibroblasts. Three full-thickness wounds were created on the backs of guinea pigs and an acellular collagen sponge, a collagen sponge seeded with autologous fibroblasts, and a collagen sponge seeded with allogeneic fibroblasts were transplanted. Before implantation, fibroblasts were labeled with PKH26. The guinea pigs were sacrificed 1, 2, and 3 weeks after implantation. The epithelization and contraction of the wounds were assessed, and the viability and distribution of the seeded fibroblasts were observed in cross sections.

Results

Three weeks after implantation, the PKH26-labeled autologous and allogeneic fibroblasts remained viable. In the wounds covered with the autologous fibroblast-seeded collagen sponge, the epithelization was fastest, and the percent wound contraction was smallest. In contrast, in the wounds covered with allogeneic fibroblasts, the epithelization was slowest and the percent contraction was largest.

Conclusion

The allogeneic fibroblasts seeded in the collagen sponge survived and remained viable on the grafted area, but did not accelerate wound healing.

Introduction

We developed a bilayered acellular artificial skin (artificial dermis) composed of an upper silicone sheet and a lower collagen sponge [1, 2, 3, 4, 5] by modifying the material described by Yannas and Burke [6, 7]. When the artificial dermis is implanted into a full-thickness skin defect, fibroblasts infiltrate the artificial dermis, and the formation of new capillaries and the deposition of extracellular matrix are observed in the collagen sponge. The collagen sponge is biodegraded and dermis-like tissue is formed in 2 or 3 weeks. To shorten this period, we seeded fibroblasts into the sponge and cultured them in vitro to produce cultured artificial dermis before implantation. In our previous study, when fibroblasts were cultured in a collagen sponge, the pores became filled with secreted dermal extracellular matrix in 4 weeks [8].

Various types of cultured dermal substitute have been reported [9, 10, 11, 12, 13, 14]. Collagen gel, collagen/GAG matrix, spongy collagen matrix, and synthetic biodegradable matrix are used as scaffolds and seeded with autologous or allogeneic fibroblasts. The autologous and allogeneic cultured dermal substitutes have been used for clinical application and are reported to improve wound healing and reduce wound contraction [10, 11, 12].

Nevertheless, the viability and distribution of autologous and allogeneic fibroblasts seeded on dermal substitutes after implantation have not been reported and the usefulness of the seeded fibroblasts is still unclear. This is probably because it is difficult to distinguish seeded fibroblasts from infiltrated cells in vivo. In this study, we created full-thickness wounds on the backs of guinea pigs and applied an acellular collagen sponge and collagen sponges seeded with autologous or allogeneic fibroblasts labeled with fluorescent membrane dye. Then we evaluated the tissue regeneration and wounds based on the viability and distribution of the seeded fibroblasts.

Section snippets

Materials and methods

Animal experiments were carried out according to a protocol approved by the Animal Experimental Committee of Kyoto University Graduate School of Medicine.

In vitro culture

After 3 weeks of culturing in vitro, fibroblasts attached to the collagen fibers were PKH26-positive (Fig. 1A, B). Fibroblasts attached to the culture dish were also PKH-positive (Fig. 1C, D). PKH-labeled fibroblasts were thus detectable and viable after 3 weeks.

Evaluation of epithelized area

The appearance of the skin defects is shown in Fig. 2. One week after implantation, granulation was formed in the wound where the autologous dermal substitutes had been implanted. Two weeks after implantation, the wound covered with

Discussion

Cultured dermal substitutes with allogeneic fibroblasts have been reported to improve wound healing and are used clinically [10, 11, 12, 13, 14, 20, 21]. To produce autologous cultured dermal substitutes, we must obtain a skin sample from the patient. It takes several weeks to isolate and culture fibroblasts, and it is impossible to use autologous cells for emergency use. Therefore, allogeneic cultured substitutes are used for the treatment of skin defects or skin ulcers and some of them have

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Wound healing/Plastic surgeryViability and Function of Autologous and Allogeneic Fibroblasts Seeded in Dermal Substitutes after Implantation

Background

Materials and methods

Results

Conclusion

Introduction

Section snippets

Materials and methods

In vitro culture

Evaluation of epithelized area

Discussion

Biomaterials

Br. J. Plast. Surg

Br. J. Plast. Surg

Br. J. Plast. Surg

Methods Cell Biol

J. Inv. Dermat

J. Invest. Dermatol

J. Invest. Dermatol

Biomaterials

Review of acellular and cellular artificial skins

Tissue Engineering

Design of an artificial skin I. Basic design principles

J. Biomed. Mater. Res

Design of an artificial skin II. Control of chemical composition

J. Biomed. Mater. Res

A new skin equivalentKeratinocytes proliferated and differentiated on collagen sponge containing fibroblasts

Plast. Reconst. Surg

Living tissue formed in vitro and accepted as skin-equivalent tissue of full thickness

Science

Tissue-engineered productAllogeneic cultured dermal substitute composed of spongy collagen with fibroblasts

Artif. Organs

Allogeneic skin substitutes applied to burn patients

Burns

Wound healing/Plastic surgery
Viability and Function of Autologous and Allogeneic Fibroblasts Seeded in Dermal Substitutes after Implantation