Fisher John P. e.a. (ed.) Tissue Engineering

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(b)(a)

(d)(c)

FIGURE 18.1 Histological comparison of native skin and cultured skin substitute prepared for treatment of pediatric

burn patient. (a) Native human skin. (b) Cultured skin substitute in vitro, shown after 6 days of incubation. Graft

was transplanted to patient after 10 days of in vitro incubation. (c),(d) Healed autograft (c), and healed cultured skin

substitute (d), biopsied 3 weeks after grafting to excised full-thickness burn wounds. Scale bars = 0.1 mm.

has been in the treatment of patients with burns affecting greater than 50% TBSA. In these patients, donor

sites for autografting are extremely limited and adjunctive treatments for wound coverage are required.

For preparation of CSS, primary cultures of keratinocytes and ﬁbroblasts are isolated using standard

techniques from a small split-thickness skin biopsy that is usually taken during a patient’s ﬁrst autograft-

ing procedure [26,27,71,72,78,79]. Selective in vitro culture of keratinocytes and ﬁbroblasts stimulates

exponential increases in cell numbers, resulting in very large populations of cells in only 2 to 3 weeks

of culture [78]. Grafting to patients can generally be performed within 2 weeks of inoculation of CSS,

which corresponds to 4 to 5 weeks after the initial patient biopsy. Because the CSS contains both dermal

and epidermal layers and develops a functional basement membrane in vitro, grafting of CSS can replace

both skin layers in a single surgical procedure (Figure 18.1) [80]. Culture of CSS at the air–liquid interface

promotes development of a stratiﬁed epidermal layer with functional barrier properties in vitro, providing

protection of the wound immediately after grafting [68,80,81]. CSS have been used with favorable results

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Skin Substitutes 18-7

as an adjunctive treatment for the healing of large burn wounds, and have been shown to signiﬁcantly

reduce the requirement for autograft and shorten the number of surgical procedures needed for deﬁnitive

wound closure [27,67,68]. CSS have also been used to a limited extent for treatment of chronic wounds

and congenital giant nevi [28,29].

18.4 Clinical Considerations

Multiple clinical factors can determine whether treatment of wounds with engineered skin substitutes

will result in skin repair. Modiﬁcations of care protocols for wounds must be used to compensate for the

anatomic and physiologic deﬁciencies in engineered skin. Currently available skin substitutes are avascular,

tend to heal more slowly than skin autograft, and may be mechanically fragile. Factors that affect the clinical

outcome with bioengineered skin replacements include, but are not limited to: composition at the time

of grafting; wound bed preparation; control of microbial contamination; dressings and nursing care; and

survival of transplanted cells during vascularization of grafts.

Attachment of cultured epithelium to a dermal substitute in vitro is advantageous because both epi-

dermal and dermal components can be applied in a single procedure, similar to skin autograft. Culture

conditions can be optimized to promote deposition of basement membrane proteins at the dermal–

epidermal junction prior to grafting, thereby eliminating the problem of blistering that is frequently

observed after grafting of epithelial sheets [33,80]. Alternatively, dermal and epidermal components of

skin substitutes may be applied in two stages: ﬁrst, application of a dermal substitute followed by vascu-

larization; and second, grafting of an autologous epidermal substitute [45,82,83]. This two-step approach

increases the density of blood vessels and extracellular matrix in the wound bed, and has been reported

to improve engraftment of cultured keratinocyte sheets. However, it requires two surgical procedures to

achieve permanent wound closure.

The lack of a vascular plexus is a major limitation of all skin replacements currently available. Split-

thickness skin contains a vascular plexus and adheres to debrided wounds by coagulum. Inosculation of

vessels in the graft to vessels in the wound occurs within 2 to 3 days [84]. In the absence of microbial

contamination or mechanical damage, autograft skin is generally engrafted and reperfused within 1 week

after transplantation. In contrast, current clinical models of engineered skin substitutes are avascular,

requiring reperfusion from de novo angiogenesis. The time required for perfusion is proportional to the

thickness of the dermal component of the skin substitute, and is longer than perfusion of split-thickness

skin. Vascularization can be accelerated by secretion of angiogenic factors from engineered skin containing

keratinocytes and ﬁbroblasts, but growth factors alone cannot compensate for the lack of a vascular plexus

prior to grafting [85,86]. The additional time required for vascularization may contribute to epithelial

loss from microbial destruction and nutrient deprivation.

Due to the delayed vascularization of skin replacements, control of contamination is critical for engraft-

ment. Topical antimicrobials are more effective for control of wound contamination than parenteral agents

[87]. Topical treatments must provide effective coverage of a broad spectrum of microorganisms, but must

have low cytotoxicity to allow healing to proceed. It is also important to avoid overlap of topical agents

with parenteral drugs used for treatment of sepsis, which could facilitate development of resistant organ-

isms. Several studies have identiﬁed individual agents, and mixtures of multiple agents, which are effective

against common wound organisms but are not inhibitory to proliferation of keratinocytes and ﬁbroblasts

[88–90].

An additional limitation of engineered skin substitutes is mechanical fragility, which contributes to

graft failure due to shear and maceration. For delicate grafts, a backing material can facilitate handling

and attachment to the wound. For example, CEA are routinely attached to petrolatum-impregnated gauze

for surgical application [91]. However, this material may not be compatible with wet dressings used to

manage infection. CSS may be handled and stapled to wounds with a backing of N-Terface™, a relatively

strong, nonadherent, highly porous material [25,26]. Porous dressings do not interfere with the delivery

of topical solutions and permit drainage of wound exudate.

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18-8 Tissue Engineering

An important practical obstacle to the routine clinical use of skin substitutes, as with any engineered

tissue, remains the high cost of their preparation and care. Estimates for the cost of keratinocyte sheets

range from $1,000 to $13,000 for each percent of absolute TBSA [63,92]. If a dermal substitute is also

included, the cost can be expected to approximately double [8,45]. Therefore, expense can become

a limiting factor for treatment of very large wounds, such as those seen in severely burned patients.

Unfortunately, these are the patients most in need of skin substitutes. Although the use of skin substitutes

can theoretically reduce the number of surgeries required to heal large burns, which should decrease the

total time of hospitalization, there are currently no studies that clearly demonstrate a decrease in costs by

use of skin substitutes of any kind. Engineered skin grafts remain an important adjunct to conventional

skin grafting, particularly in the treatment of burns, but cannot be used as a primary modality of wound

closure except in the most extreme cases [61].

18.5 Assessment

The outcome after treatment of wounds with bioengineered skin substitutes must be measured to determ-

ine whether the beneﬁts justify any risks associated with the therapy. Qualitative outcome, which relies

heavily on the trained eye of the clinician, can be assessed through clinical evaluation integrating multiple

properties of the wound. For example, the Vancouver Scale is used for assessment of burn scar by trained

clinicians, and provides an ordinal score for properties of skin including pigmentation, vascularity, pli-

ability, and scar height [93]. Such scales assign quantitative values to qualitative measurements and can

provide a relative comparison for evaluation, but they are inherently subjective and dependent on the

examiner.

Objectivity may be increased by use of noninvasive instruments to measure biophysical properties

of skin, including vascular perfusion, epidermal barrier, pliability, color, and surface pH. Quantitative

assessment of skin substitutes can highlight deﬁciencies compared to normal skin or split-thickness

autograft, or can be used to assess the advantage of skin substitutes to the patient without interfering

with recovery. Although no single biophysical property is deﬁnitive, multiple measurements can provide a

general assessment for evaluation of outcome. For example, measurements of surface electrical capacitance

(SEC) can be used to deﬁne the degree of skin barrier development [94]. SEC is measured using a dermal

phase meter, an instrument that is easily used in a clinical setting, with minimal pain or discomfort for the

patient [95]. Pigmentation of grafted wounds treated with engineered skin substitutes can be measured

using a chromameter. Multiple parameters of skin function must be measured to quantify overall beneﬁt

from treatment with skin substitutes.

18.6 Regulatory Issues

In the United States, it is the responsibility of the Food and Drug Administration (FDA) to protect the

public from health risks associated with new medical therapies. FDA approval requires that new therapies

be safe and effective, and that the probable beneﬁts to health outweigh the probable risks of the therapy

or of the untreated disease or condition [96]. Safety considerations for engineered skin substitutes must

take several factors into account, including media composition, tissue acquisition, graft fabrication and

storage, and sterility testing of the ﬁnal product [96]. For example, cell culture media must be of the

highest purity and free from toxic contaminants. Cells derived from allogeneic donors must test negative

for transmissible pathogens. Autologous cells must be handled carefully as well. Because autologous tissues

are not routinely screened for pathogens, universal precautions to protect laboratory personnel must be

practiced. Xenogeneic components, such as bovine collagen, must not only be free from pathogens that

can cross species boundaries, but must also be nonimmunogenic. If xenogeneic cells, such as irradiated

3T3 mouse ﬁbroblasts, are used to facilitate initiation of keratinocytes cultures, compliance with safety

standards for xenogeneic transplantation must be assured [97]. Although these common cells are generally

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thought to be free from risk of disease transmission, unknown risks may exist, and hence patients are

excluded from future donation of blood or body parts [36,98].

Bioengineered skin substitutes may be regulated as either devices or biologics, depending on their

composition and “primary mode of action.” Skin substitutes consisting of autologous cells only, or an

acellular human tissue matrix, may not require collection of effectiveness data for regulatory approval.

Living autologous cell populations intended for structural repair are consideredto be inherently efﬁcacious

[99]. However, if no effectiveness data are collected, no claims of effectivenesscan be made. Skin substitutes

that combine cells with biopolymers are currently considered class III (signiﬁcant risk) devices that require

demonstration of effectiveness in addition to safety [96].

18.7 Future Directions

Despite encouraging clinical results with bioengineered skin for the adjunctive treatment of burns, chronic

wounds, and other skin deﬁciencies, skin substitutes containing just two cell types are limited by anatomic

and physiologic deﬁciencies compared to split-thickness skin autograft. Several areas of preclinical invest-

igation suggest that skin substitutes can be further engineered to increase homology to native human skin.

These include the incorporation of additional cell types to improve functional and cosmetic outcome, and

the use of genetically modiﬁed skin cells to enhance performance after grafting.

18.7.1 Pigmentation

Normal skin pigmentation results from the appropriate epidermal distribution and function of melano-

cytes. The most critical function of melanocytes is protection from ultraviolet irradiation, but they have

psychological importance as well, as a patient’s body image and personal identity can impact recovery from

massive skin injury [100–102]. Pigmentation of cultured skin may result from transplantation of “passen-

ger” melanocytes, which may persist in selective cultures of epidermal keratinocytes [67,79]. Melanocytes

can survive under conditions used for keratinocyte culture, though they proliferate at slower rates and are

depleted upon serial passage or cryopreservation [103–105]. In CSS grafted to excised burns, pigmented

areas resulting from passenger melanocytes have been observed as individual foci within two months after

transplantation [79]. By 1 to 2 years after healing, the foci increase in area, occasionally fusing together

to form larger pigmented regions. Uniform pigmentation was demonstrated in preclinical studies with

CSS deliberately populated with selectively cultured human melanocytes [106–108]. Future studies will

be needed to address regulation of the level of pigmentation in uniformly pigmented cultured skin.

18.7.2 In Vitro Angiogenesis

The absence of a vascular plexus in bioengineered skin necessitates vascularization to occur de novo, rather

than through inosculation of the graft with the wound, increasing the time of nutrient deprivation and

susceptibility to microbial contamination after grafting. This limitation can be indirectly addressed in a

clinical setting by irrigating the graft with solutions of nutrients and antimicrobial agents for several days

after transplantation [27,67,109]. A direct approach would be to initiate angiogenesis in the skin substitutes

in vitro, prior to grafting. This would permit vascularization to occur through both inosculation of existing

vessels and also neovascularization, as occurs for grafted split-thickness skin [84].

Initiation of angiogenesis in vitro requires the addition of endothelial cells to the engineered skin.

Endothelial cells may organize into vascular structures in culture with the aid of biomaterial supports and

coculture with accessory cells. For example, engineered blood vessels have been constructed in vitro using

mixed cultures of ﬁbroblasts, human umbilical vein endothelial cells (HUVEC), and vascular smooth

muscle cells in a collagen matrix [110]. In preclinical studies, transplantation of engineered blood vessels

constructed by culture of HUVEC in three-dimensional collagen/ﬁbronectin gels has been reported [111].

More recently, a composite cultured skin containing HUVEC and keratinocytes in a human dermal matrix

was reported, which displayed evidence of perfusion after grafting to mice [112]. These studies illustrate

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18-10 Tissue Engineering

the feasibility of grafting synthetic vessels in cultured skin, but overexpression of Bcl-2 through retroviral

modiﬁcation was required to promote survival of the transplanted endothelial cells [111]. In addition,

another potential limitation of these studies that will impede their clinical application is the reliance on

nondermal or nonautologous endothelial cells. Ideally, multiple cell types (keratinocytes, ﬁbroblasts, and

human dermal microvascular endothelial cells, or HDMEC) could be derived from a single autologous skin

sample. Transplantation of HDMEC in a composite skin substitute containing isogenic keratinocytes and

ﬁbroblasts was demonstrated in an athymic mouse model, though perfusion was not observed [113]. The

transplantation of HDMEC in a clinically relevant cultured skin model showed the feasibility of preparing

autologous cultured skin containing HDMEC, but future studies must demonstrate inosculation of vessels

in the graft with vessels in the wound bed to yield improved performance after grafting.

18.7.3 Cutaneous Gene Therapy

Keratinocytes and ﬁbroblasts are amenable to genetic modiﬁcation in vitro by a variety of methods.

Genetically modiﬁed cells can be used to populate engineered skin substitutes. This is termed “ex vivo”

gene therapy because cells are removed from the body and genetically modiﬁed in culture before being

transplanted back to the recipient. There has been a great deal of interest in the use of genetically modi-

ﬁed skin substitutes for the treatment of cutaneous diseases. For example, preclinical studies suggest that

ex vivo gene therapy can be useful for treatment of lamellar ichthyosis, a condition characterized by defect-

ive epidermal barrier, and the blistering skin disease junctional epidermolysis bullosa (JEB) [114–116].

Theoretically, genetically modiﬁed keratinocytes can be transplanted for secretion of circulating factors

to treat systemic diseases. Keratinocytes have been genetically modiﬁed to secrete human growth hor-

mone and clotting factor IX, but therapeutic protein levels have been difﬁcult to obtain after grafting

[117–121].

Another application of cutaneous gene therapy is the regulation of wound healing. Hypothetically,

genetic modiﬁcation may be used to overcome anatomic limitations or to enhance their biological activ-

ity. For example, keratinocytes modiﬁed to overexpress the mesenchymal cell mitogen Platelet Derived

Growth Factor-A (PGDF-A), seeded on an acellular dermal matrix, showed increased cellularity, vascu-

larization, and collagen deposition after grafting to mice, suggesting improved function due to PDGF-A

overexpression [122]. In other studies, keratinocytes were genetically modiﬁed by retroviral transduc-

tion to overexpress the angiogenic cytokine Vascular Endothelial Growth Factor (VEGF) [85,86]. After

transplantation to athymic mice, skin substitutes containing ﬁbroblasts and VEGF-modiﬁed keratinocytes

showed enhanced and accelerated vascularization, decreased contraction, and increased engraftment com-

pared to control grafts containing unmodiﬁed cells [85,86]. Thus, genetic modiﬁcation of keratinocytes

can hypothetically be used to overcome the lack of a vascular plexus in engineered skin grafts.

A particularly promising avenue of research involves the genetic modiﬁcation of cells in cultured skin

grafts to reduce or eliminate immune rejection. Preclinical studies have shown that reduced expression

of major histocompatibility complex (MHC) class I and II antigens can prolong engraftment of skin

grafts in mouse allograft models [123]. In one study, fetal skin was used because it exhibited substantially

reduced MHC class I and II expression compared with neonatal skin. In another study, keratinocytes

were genetically modiﬁed to overexpress indoleamine 2,3-deoxygenase (IDO), a tryptophan-catalyzing

enzyme that functions to prevent fetal rejection during pregnancy [124]. Increased IDO expression in

keratinocytes led to a down-regulation of MHC class I expression. These studies suggest a possible

mechanism for preparation of allogeneic skin substitutes that would escape immune rejection, and may

someday lead to universal donor cultured skin grafts.

18.8 Conclusions

Technological advances in the fabrication of biomaterials and the culture of skin cells have permitted

the production of bioengineered skin substitutes. These have provided improved therapeutic options for

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Skin Substitutes 18-11

patients suffering from acute or chronic wounds, and offer the promise of new treatments for inherited

cutaneous diseases. Continued research will be needed to identify more efﬁcient methods to utilize pre-

cious autologous tissue, which will provide greater amounts of skin substitutes for grafting as well as

shorten the time required for their preparation. Increasing the complexity of skin substitutes, from acellu-

lar biopolymers to composite materials with multiple cell types, will result in continued improvements in

anatomy and physiology, working toward greater homology to native human skin. These improvements

will lead to enhanced performance of engineered skin grafts, greater clinical efﬁcacy, and reduction of

morbidity and mortality for patients with wounds or cutaneous disease.

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