Recipes for Reconstituting Skin

E. Bell M. Rosenberg

P. Kemp II. Gay G. D. Green N. Muthukumaran C. Nolte Organogenesis Inc., Cambridge, Massachusetts 02142

Reconstituted Living Skin Equivalent™ (LSE™) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilayered differentiated epidermis. The dermal equivalent develops through interactions between fibroblasts and collagen fibrils that begin to form after the cell-matrix precursor is cast. The gel that forms as a result of collagen polymerization and fluid trapping is contracted uniformly in all dimensions. By securing it at ends and edges in the mold in which it is cast, the final dimensions, strength and morphology of the forming tissue are altered. The same phenomena are seen in casting tubular tissues for the fabrication of small caliber blood vessel equivalents. The cells of the dermal equivalent are biosynthetically active and enrich the matrix to different degrees with secretory products, depending on how the cells are stimulated and on the presence or absence of an epidermis. Collagen biosynthesis by dermal cells in the DE is sensitive to growth factors, ascorbate concentrations and amino acid pools. Both ascorbate and TGF[5\ increase total collagen biosynthesis at least two-fold by one week after tissue formation. With TGFfii present, the capacity of cells in the DE to synthesize collagen increases with time, over a two-week period. If ascorbate (200 fig/ml) is added just after the tissue is cast and daily thereafter, contraction lattice is blocked, and collagen biosynthesis is enhanced relative to contracted controls that had received 200 ng/ml ascorbate once. The increase was nearly an order of magnitude over that of controls and was coordinate with a comparable increase in hyaluronate and sulfated glycosaminoglycan (GAG) production as shown by TCA-precipitable glucosamine in the intercellular matrix of the DE. Both the LSE and the Living Dermal Equivalent™ (LDE™) exhibit complex responses to UV radiation and to various chemicals that are greatly different from responses given by monolayered cells. In general, threshold doses are elevated by one or more orders of magnitude for the tissues as compared with cells in monolayer, with the LSE exhibiting higher thresholds than the DE. The immunogenicity of the human LSE has been tested in vitro. Its cells are shown to be unable to stimulate a response in a mixed lymphocyte reaction (MLR) even after Class II antigens are induced by exposure to cytokines. The basis for the immunologic neutrality of the LSE can be referred to the absence of immune system (IS) cells normally present in skin and to the specific antigenic profiles of nonimmune system (NIS) cells that must be different from those of IS cells and which, even after Class II induction, are not allostimulatory. The generality of immunologic neutrality is an essential consideration in the fabrication of tissue and organ equivalents for grafting. The idea that it can be made a graft property has been formalized in the Neutral Allograft Hypothesis.

Fabricating Tissues in Three Dimensions The title of our paper, Recipes for Reconstituting Skin, is meant to encompass a review of work in the laboratory at M.I.T. and in the laboratories at Organogenesis Inc. on the reconstitution of tissue and organ equivalents from specialized cultivated cells and extracellular matrix molecules. Initially, reconstituting tissues and organ equivalents was inspired by their potential usefulness as model systems for studying cell differentiation and cell physiology under conditions more closely resembling those found in vivo. While that objective remains, especially because of growing requirements for living in vitro test systems in the realm of applied research, engiContributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICAL ENQINEERING. Manuscript received by the Bioengineering Division January 20, 1991; revised manuscript received February 1, 1991.

neering tissues for use as grafts to replace diseased, damaged, or aged body parts has also become a rapidly growing activity. In organisms, tissues are constituted in several ways: some connective tissues, like dermis, cartilage, bone, smooth muscle, corneal stroma, consist of cells surrounded by variable amounts of matrix. The secreted matrix components undergo some selfassembly and are organized by a differentiated cell population that defines the tissue. The character of the matrix is determined by the phenotype of the tissue cell. Distinguished from the foregoing cell-matrix assemblies are epithelial tissues in which cells are not separated by intercellular matrix but are in direct contact with one another. Epithelia may be simple, that is monolayered, including endothelia of blood vessels and mesothelia that line the pleural and peritoneal cavities, as well as intestinal, bronchial and other epithelia. Epithelia may also be MAY 1991, Vol. 113/113

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Fig. 1 Medial layer 01 a blood vessel equivalent cast In a special cham· ber. Smooth muscle cells combined with collagen and nutrient medium are Introduced Into the chamber around a central core or mandrel. A collagen·cell lattice lorms (top photo) and Is condensed by the smooth muscle cells radially with Iluid being expressed Irom the lattice (lower photo). An adventitial layer can be applied when the medial layer Is contracted, aller removal 01 the expressed fluid. Note that longitudinal contraction of the tube that lorms Is prevented by restraining Its ends. By extracting the mandrel, It becomes possible to seed the luminal surface 01 the media with endothelial cells Introduced as a suspension. By rotating the tube slowly, cells will settle on Ihe surface and attach to It [2J.

stratified, such as in esophageal, tracheal, or epidermal tissues. The foregoing epithelia exists as sheets that cover surfaces. In addition, glandular epithelia in the form of tubes, cords, or acini can be organized as discrete structures surrounded by cell containing matrix tissues. As a rule, at the interface between epithelia and matrix tissues, the epithelia lay down an acellular strip of matrix usually called a basal lamina. We have found that matrix tissue equivalents, that is tissues in which cells are separated by an intercellular matrix, can be cast in a mold by combining cells, nutrients and matrix components, mainly collagen, under conditions that lead to formation of a gel. The gel forms as collagen polymerizes into a random lattice of native banded fibrils in which fluid is trapped. Connective tissue cells, such as fibroblasts, adipocytes, muscle cells, tendon and ligament cells, as well as other cell types, make contact with the fibrils of collagen, attach to them and compact them by drawing the fibrils toward themselves as attached cell podia contract. The contractile activity of the cells causes fluid to be expressed from the lattice, so that its volume diminishes [l]. As a result, the cell density per unit volume, as well as the collagen density, increase. The dimensionality of the volume reaction that occurs can be regulated by imposing restraints on the contracting lattice. If the perimeter of a rectangle is fixed, the contraction will occur in the thickness mode only, even though the cells are in traction along lines perpendicular to the thickness axis. In casting a tube, destined to become a blood vessel equivalent, around a rodshaped core, if the ends of the gelled cell-matrix mixture are fixed, contraction is limited to the radial dimension (Fig. 1). That is not to say that the lines of stress are hoop-shaped; they are not: the cells and the collagen fibrils of the matrix structure are aligned longitudinally in the axis of the core. When such a tube is stressed by application of an internal hydrostatic force, failure is always seen as a longitudinal split. Molds have been devised to cast sheets, tubes, tori, hemispheres, spheres, and other shapes. Cells engaged in the organization of the tissue equivalent are distributed more or less uniformly through the tissue. The geometry of tissue equivalent structures resulting from the contractile activity of cells reflects 114JVo1.113, MAY 1991

Fig. 2 living skin equivalent 14 days after exposure of the epidermis to air. Below the stratum corneum (SC), there Is a granular layer (Gl) In which cells are laden with keratohyalin granules. Well·developed spl· nous (Sl). suprabasal (SBl) and basal layers (Bl) are seen In descending order. Below the epidermis Is the dermal equivalent which can be pre· pared with or without an epidermis. The overall thickness of the lSE is about 0.7 to 1.0 mm, with the epidermis measuring 0.1-0.2 mm. Bar '" 100 microns.

the geometry of the mold in which they are cast and the restraints imposed on the gel. In matrices, cells, such as fibroblasts, preadipocytes, or muscle cells, are spindle-shaped and become polarized in the matrix, i.e., oriented longitudinally along the lines of force that develop as they remodel the tissue equivalent. As expected, orientation will arise also in relation to the gel anchors against which the cells must pull. The volume reduction of the starting gel depends on the number of cells, the concentration of collagen, time and temperature. For example, at equilibrium, that is after 4 days, with 2.5 x 104 dermal fibroblasts/ml and 0.68 mg/ml of collagen, volume reduction may be between 30- and 50-fold, depending on the restraints imposed. If contraction is unrestrained, the lower value is approached; if the periphery of a disk-shaped or rectangular gel is restrained, the volume reduction approaches the higher value. Well-contracted cell-matrix tissue equivalents have been formed with dermal fibroblasts [1], vascular fibroblasts [2], astrocytes, keratinocytes [3], adipocytes [4], smooth [2], striated and cardiac muscle cells, chondrocytes, osteoblasts, macrophages and other cell types. On the other hand, glandular epithelia [5], lymphocytes or mast cells, for example, do not reduce gel volume. Keratinocytes, which have the capacity to spread as sheets, can contract gels. but not as well as cells that are normally surrounded by matrix. A contracted tissue equivalent serves as a good substrate for epithelial cells. Keratinocytes [3], vascular endothelial cells [2], intestinal cells, airway cells, corneal endothelial and epithelial cells [6] have been plated on the surfaces of contracted tissue equivalents and have been shown to differentiate well. The combined tissues constitute organotypic systems. Such systems can be variously configured. For example, the contracted dermal tissue equivalent substrate can be in contact with a nutrient reservoir of liquid or agarose placed below it [7] while the epithelial tissue made up of keratinocytes can be exposed to air, often a necessary condition for complete differentiation.

The Living Skin Equivalent The in vitro coculture of tissues that normally are contiguous in vivo and depend on one another for nutrients, differentiation signals and responses to external stimuli can come to resemble fairly closely, their actual in vivo counterparts. The LSE is such an organotypic model system (Fig. 2). The degree to which the epidermis of the LSE, that is an epithelialized cell-matrix Transactions of the ASME

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tissue, develops in vitro has been described in detail elsewhere [8]. In brief, the full morphological and biochemical range of differentiation markers typical of actual epidermis is expressed. The low molecular weight and high molecular weight keratins, involucrin and filaggrin appear in their appropriate strata. Deposited laminin, type IV collagen and {34 integrin are seen as products of a well-organized cuboidal layer of basal cells at the interface between epidermis and dermal equivalent. In progressively differentiated layers approaching the stratum

Flg.3 Transmission electron micrograph of type I collagen fibrils within the dermal equivalent stained with cupromeronlc blue. A periodic as· soclallon of proteoglycan·dye complexes can be seen along the length 01 the fibril (arrows). Living skin equivalents were stained overnight In a solution of 2.5 percent glutaraldehyde and 0.05 percent cupromeronic blue in 25 mM sodium acetate buller containing 0.3 M magnesium chlo· ride (pH 5.6). Specimens were then dehydrated In a graded ethanol series and embedded In Spurr'S resin. Sections (70 nm) were stained with 0.5 percent sodium tungstate In 50 percent ethanol. Bar = 0.2 microns.

corneum are seen granular intracellular precursors of extruded lipid lamellae that occur intercellularly in the stratum corneum

Pl.

Necessary conditions for the full development and differentiation of an epidermis arising from a plating of keratinocytes include the living contracted cellular dermal equivalent on which it grows and culture routines that provide for medium changes with the timed addition of Ca + + , and the timed exposure of the epidermis to air [8]. Development encompasses a cell division phase followed by regulated differentiation. The dermal equivalent of the LSE is remodeled by the fibroblasts that occupy it. By compacting the collagen fibrils in the lattice, the concentration of collagen per unit volume is increased 30- to 50-fold. As the lattice becomes condensed, fibrillogenesis is enhanced; fibrillar components become more concentrated in the vicinity of cells than elsewhere, and they are also organized by cells into distinct patterns. Evidence of differentiation in the dermal equivalent is seen in the extracellular matrix into which the dermal fibroblasts secrete processed collagen [9] and other products. Dermatan sulfate proteoglycan is not a constituent of the starting matrix mixture combined with cells when the dermal equivalent is cast, thus its presence in the matrix 14 days after the dermal equivalent is cast suggests that it is a product of cells in the lattice. Its presence has been confirmed by cellulose acetate electrophoresis and alcian blue staining. Localization has been demonstrated by staining the matrix with cupromeronic blue [l0] (Fig. 3) which shows the glycosaminoglycan (GAG) localized along collagen fibrils at the site of the d bands. Hyaluronic acid is also synthesized by dermal equivalent cells (Fig. 4). The art of medium supplementation and optimization is one that can be practiced in the reconstitution of complex tissue structures just as it has been in cultivating cells on monolayers. As with monolayers, designing media for tissue equivalents, has as a goal stimulation of cell division, but it is only a

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Fig. 4 Biosynthesis Under Various Conditions of Extracellular Matrix Constituents by Fibroblasts in Dermal Equivalents Expressed as Percent of Control, Normalized to Cell Number: Human foreskin dermal fibroblasts were mixed (25,OOO/ml) with a nutrient medium (Dulbecco's Modified Eagle's Medium [DMEMl, 10 percent newborn calf serum, 20 mM l· glutamine, and 50 I'g/ml gentamicin sulfate) plus 0.67 mg/ml bovine type I collagen and allowed to set in 0.5 ml volumes in 48·well plates without tissue culture pretreatment. After 24 hours of incubation at 37° under 10 percent CO2 , dermal equivalents were sep· arated from well walls by rimming. At this time, 0.5 ml of nutrient medium was added containing 200 I'g/ml sodium ascorbate ("A200"), 1.0 mM/l·glycine t'G"), 1.0 mM L·proline (UP"), 1.0 mM L·serine (US"), bringing the final volume of each sample to 1.0 mi. After further incubation, 10 I'Ci of l.[5-3H\',r.roline (15 CllmMole), 10 I'Ci of D.[6. 3H(N)!.glucosa· mine-HCI (27 CilmMole), or 5 I'CI of S·sodium sulfate (541.3 mCllmMole) were added to the medium at 7 days after casting; incorporation of radlolabel into extracellular matrix was analyzed 9 days after casting. Six dermal equivalents per condition were separately extracted with 500 1'1 of 1 M acetic acid, of which 50 1'1 was precipitated with 20 percent trichloroacetic acid. Precipitates were resuspended and measured by scintillation count· ing. The remainder of each 3H.proline-labeled extract was hydrolyzed in 6 N HCI (150°C) and derivatized with phenl,lisothiocyanate (PITC) before reverse-phase HPLC separation of 3H·hydroxyproline from H·proline. Scintillation counting of the eluted 3H.hydroxyproline provided data for measurement of collagen biosynthesis.

Journal of Biomechanical Engineering

MAY 1991, Vol. 113 J 115

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Recipes for reconstituting skin.

Reconstituted Living Skin Equivalent (LSE) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilay...
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