Microgravity Science & Applications Program Tasks and Bibliography for FY 1996 Details

MSAD Program Tasks - Ground-based Discipline: Biotechnology

The Effect of Microgravity on the Human Skin Equivalent

Principal Investigator: Dr. S. D. Dimitrijevich Univ. of N. Texas Health Science Ctr, Fort Worth

Co-Investigators:
Mills, D.O. University of North Texas Health Science Center @ Fort

Task Objective:

The objective of this project in the first instance is to effect morphogenesis of The Human Skin Equivalent under the conditions of simulated microgravity and compare its tissue characteristic properties with those of the Skin Equivalent generated under 1g conditions. We will then study the effects of fluctuating levels in several physiological parameters (e.g., glucose and calcium), which intact organisms experience under zero gravity, on the development and functions of our in vitro model of human skin.

Task Description:

We have developed several living in vitro models of human tissue (e.g., skin, cornea, conjuctiva), based on a non-contracting three-dimensional collagenous matrix populated with appropriate fibroblasts. Because these models support functional epithella and/or endothelia and closely mimic ex vivo human tissue, they provide an excellent opportunity for studies of cell-cell and cell-matrix interactions during tissue development or repair. Of equal importance are the responses of these models to changes in their microenvironment and return to the quiescent state. We will, therefore, examine all stages of development of the skin equivalent ranging from fibroblast behavior and function, attachment of keratinocytes and melanocytes, and epidermopoesis under the conditions of simulated microgravity. We also intend to study the effect of changes in glucose and calcium homeostasis, transiently occurring under zero gravity in man, on the Human Skin Equivalent cultured in RWV. Finally, we propose to determine if the exposure of tissue to simulated microgravity represents a stress to which cellular components respond by expression of stress proteins.

Task Significance:

Simulation of the microgravity environment using RWV has been found to be a unique environment in which cells which are otherwise difficult to grow may be successfully cultured. Furthermore, under these conditions, a number of cells form large aggregates. We hope to demonstrate that the simulated microgravity environment is also conducive to generation of human tissue, and that such tissue will possess biological and mechanical integrity appropriate for use in tissue replacement therapy. We further hope to increase our understanding of tissue tolerance to the transient physiological abnormalities experienced under zero gravity, and enhance our understanding of how these might impact tissue development and repair and contribute to the design of new approaches to wound healing.

Progress During FY 1996:

Background Our interest in cell-matrix interactions has led to the development of three-dimensional models based on non-contracted collagen type I gels inhabited by fibroblasts derived from normal human tissues. Cells populating such gels are ideally situated for gravity nullification because the "high viscosity" of the matrix restricts the centrifugal acceleration generated in the rotating-wall vessel (RWV). Thus rotation of the developing tissue around a horizontal axis at a constant angular velocity allows each component of the living system to describe a perfect circular path independent of rotational speed (Stokers' motion).
Modified Rotating-Wall Vessels (RWVs) Initially, human skin equivalent (SE) construction requires polymerization of collagen type I solution containing normal dermal human fibroblasts into a three-dimensional gel-dermal equivalent (DE). After fibroblasts have adapted to the matrix, keratinocytes (alone or mixed with melanocytes) are plated on the surface of the DE and allowed to attach and form a confluent monolayer. Differentiation of keratinocytes into a stratified cornified epidermis is then effected by raising extracellular calcium and elevation of the SE to an air-liquid interface.
Our early experiments showed that the vessel cylinder wall is too highly polished and hydrophobic to allow polymerization of collagen into a three-dimensional gel matrix. Consequently, during the past year, we have designed and evaluated two RWV formats, based on the 50 ml STLV, which satisfy our gravity nullification requirements and are suitable for the construction of the SE. One modification (M1) involved machining four segmental compartments into the rear end cap of the STLV; the other modification (M2) has four identical size compartments machined into the vessel cylinder. In M1, DE, are cast simultaneously in the four compartments (generating segments of tissue), whereas in M2, DEs are cast in each compartment individually to produce rectangular pieces of tissue. Using either vessel, one RWV culture set up is used to generate four DE/SE under identical conditions. Since the experimental protocols involved in SE development require a minimum of 3 weeks, our modified RWVs also optimize hardware utilization.
Dermal Equivalents Generated in RWV Fibroblast Phenotype: We have shown that the weight, volume, and therefore, density of DEs generated in 1g are independent of collagen type I concentration (3-5mg/ml) and cell number (fibroblasts, 105 - 5 x 105 cells/ml). Under these conditions, there is also no significant cell division or migration. This in vivo -like quiescence is maintained in the DE generated under microgravity during a 15-day observation period. Since fibroblasts respond to mechanical stimuli and are responsible for tissue contraction, developing DE is very sensitive to mechanical stresses. It was particularly important to observe that DEs generated in the RWV do not contract. In addition to indicating an absence of the "activated" fibroblast phenotype, the myofibroblast, this finding also suggests that the microgravity environment does not appear to impose significant mechanical stress on the DE.
Treatment of the DE with neutral red confirmed cell viability. Paraffin-embedded, cross-sectioned DE showed a random distribution of cells throughout the matrix. In contrast, some "sedimentation" for fibroblasts is frequently observed under 1g conditions.
Confocal microscopy: A more detailed and precise assessment of fibroblast distribution, orientation, and interaction with the collagen matrix will be obtained from studies of DEs by confocal microscopy (CFM). In order to facilitate such findings, we have developed methods for labeling fibroblasts with fluorescent Cell Tracker dyes, and incorporated the labeled viable cells into DEs. The utility of this technique was demonstrated by the analysis of orientation of a single fibroblast in a DE, which was defined by eight optical sections obtained at 9m intervals. Cell-matrix interactions are highly dependent on cell surface matrix receptors (integrins) and the extracellular components present. We are currently studying the expression of collagen-specific integrins and the organization of collagen fibers in DEs developed in the RWV.
Mechanical Properties: Tensile strength of skin, primarily a feature of the dermis, is particularly important in the context of wound healing and clinical use of SEs as graft tissue. Preliminary experiments suggest that DEs generated in the RWV have a lower tangential stiffness (about 3-6 fold) than normal human dermis. Such differences in mechanical properties might be expected in view of the lower collagen fiber density in DE as shown by transmission electron microscopy.
Metabolic Activity: Cell viability in DEs cultured in 1g can be maintained for the period of time required for epidermalization of the SE. Preliminary morphological data obtained from RWV experiments suggests that fibroblasts begin to show signs of distress after about 2 weeks. Initial metabolic studies suggest that glucose depletion or lactate accumulation are probably not contributing stress factors. Since the DE is submerged under a considerable volume of medium, hypoxic conditions might develop. Increasing oxygenation of the DE will be discussed below.
Skin Equivalents Generated in RWV Construction: Although normal human keratinocytes and melanocytes are readily available in our laboratories, the initial focus is on the effect of microgravity on bicellular SE composed of fibroblasts and keratinocytes. Conditions for growth and differentiation of keratinocytes, which are also optimal for melanocyte survival, are being developed to facilitate the construction and studies of the tricellular SE.
In a typical SE construction experiment, once DEs are established, the medium (Hams F12) is removed from the RWV, keratinocytes are placed on and allowed to attach to the surface of DEs (2-3 hours). The vessel is then charged with keratinocyte growth medium (KGM) and set to rotate. When the keratinocyte monolayer is confluent, differentiation is initiated by raising the extracellular calcium. Such developing SEs, when paraffin embedded and cross-sectioned, show that differentiation through several suprabasal layers (including the granular layer) does take place. The lack of cornification, however, suggests that for complete maturation, the SE needs to be cultured at an air-liquid interface (sufficient oxygen). This is consistent with our earlier work which showed that epidermalization of the SE is stimulated by exposure to hyperbaric oxygen.
A stronger attachment of keratinocytes to the DE observed under RWV conditions indicates increased adhesion. This is manifested by increased tissue integrity at the dermal-epidermal junction observed during sectioning of embedded SEs. Expression of adhesion molecules by keratinocytes and melanocytes under microgravity is presently under study.
SE Vessel: In order to provide conditions which will allow exposure of the SE surface to air and improve oxygenation and nutrition of the DE, we have developed a third format of the RWV. In this modification (M3), the vessel is composed of two parts, each of which has its own diffuser and medium exchange parts. the "vessel divider" is composed of two discs, each containing six wells, which sandwich a semipermeable membrane. The membrane forms the "floor" of each of the six wells, providing each well with an apical and a basal side. DEs are cast in the wells and the oxygenation and nutrients are supplied from both sides. Furthermore, SEs in the wells are at the air-liquid interface when one of the vessel changers is operated with very little or no medium. We are currently evaluating this vessel format, and preliminary experiments suggest that it will satisfy the required criteria.
Confocal Microscopy of the SE: We believe that confocal microscopy will be a useful tool for studying processes involving cell-cell and cell-matrix interactions in the SEs as well as the DE. Consequently, we have shown that keratinocytes and melanocytes may be prelabeled with fluorescent Cell Tracker dyes. The fluorescence is retained in the cells through cell harvesting, seeding on DEs, proliferation, and differentiation. The SE labeled in this manner can be fixed, paraffin embedded, sectioned, and the cross-sections deparaffinized with retention of fluorescent label. Initially, this methodology will lead to the development of attachment and proliferation assays.
Technology Transfer From the inception of this project, we have worked closely with Synthecon, Inc. Their experience in engineering and manufacturing of RWVs was instrumental in generating vessels suitable for our specific tissue assembly requirements. Prototypes of M1 and M2 were displayed at the Houston Investigators Working Group meeting earlier this year. An agreement is in place for the exclusive manufacture of the modified vessels, and these should eventually be available for general use.

Students Funded Under Research: Task Initiation: 9/95 Expiration: 8/99

BS Students: 0 BS Degrees: 0 Project Identification: 962-23-01-37

MS Students: 1 MS Degrees: 0 NASA Contract No.: NAG9-813

PhD Students: 1 PhD Degrees: 0 Responsible Center: JSC

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E-Mail: peer1@hq.nasa.gov

MSAD Program Tasks - Ground-based					Discipline: Biotechnology
The Effect of Microgravity on the Human Skin Equivalent
Principal Investigator: Dr. S. D. Dimitrijevich					Univ. of N. Texas Health Science Ctr, Fort Worth
Co-Investigators:
Mills, D.O.					University of North Texas Health Science Center @ Fort





Task Objective:
The objective of this project in the first instance is to effect morphogenesis of The Human Skin Equivalent under the conditions of simulated microgravity and compare its tissue characteristic properties with those of the Skin Equivalent generated under 1g conditions. We will then study the effects of fluctuating levels in several physiological parameters (e.g., glucose and calcium), which intact organisms experience under zero gravity, on the development and functions of our in vitro model of human skin.
Task Description:
We have developed several living in vitro models of human tissue (e.g., skin, cornea, conjuctiva), based on a non-contracting three-dimensional collagenous matrix populated with appropriate fibroblasts. Because these models support functional epithella and/or endothelia and closely mimic ex vivo human tissue, they provide an excellent opportunity for studies of cell-cell and cell-matrix interactions during tissue development or repair. Of equal importance are the responses of these models to changes in their microenvironment and return to the quiescent state. We will, therefore, examine all stages of development of the skin equivalent ranging from fibroblast behavior and function, attachment of keratinocytes and melanocytes, and epidermopoesis under the conditions of simulated microgravity. We also intend to study the effect of changes in glucose and calcium homeostasis, transiently occurring under zero gravity in man, on the Human Skin Equivalent cultured in RWV. Finally, we propose to determine if the exposure of tissue to simulated microgravity represents a stress to which cellular components respond by expression of stress proteins.
Task Significance:
Simulation of the microgravity environment using RWV has been found to be a unique environment in which cells which are otherwise difficult to grow may be successfully cultured. Furthermore, under these conditions, a number of cells form large aggregates. We hope to demonstrate that the simulated microgravity environment is also conducive to generation of human tissue, and that such tissue will possess biological and mechanical integrity appropriate for use in tissue replacement therapy. We further hope to increase our understanding of tissue tolerance to the transient physiological abnormalities experienced under zero gravity, and enhance our understanding of how these might impact tissue development and repair and contribute to the design of new approaches to wound healing.
Progress During FY 1996:
Background Our interest in cell-matrix interactions has led to the development of three-dimensional models based on non-contracted collagen type I gels inhabited by fibroblasts derived from normal human tissues. Cells populating such gels are ideally situated for gravity nullification because the "high viscosity" of the matrix restricts the centrifugal acceleration generated in the rotating-wall vessel (RWV). Thus rotation of the developing tissue around a horizontal axis at a constant angular velocity allows each component of the living system to describe a perfect circular path independent of rotational speed (Stokers' motion). Modified Rotating-Wall Vessels (RWVs) Initially, human skin equivalent (SE) construction requires polymerization of collagen type I solution containing normal dermal human fibroblasts into a three-dimensional gel-dermal equivalent (DE). After fibroblasts have adapted to the matrix, keratinocytes (alone or mixed with melanocytes) are plated on the surface of the DE and allowed to attach and form a confluent monolayer. Differentiation of keratinocytes into a stratified cornified epidermis is then effected by raising extracellular calcium and elevation of the SE to an air-liquid interface. Our early experiments showed that the vessel cylinder wall is too highly polished and hydrophobic to allow polymerization of collagen into a three-dimensional gel matrix. Consequently, during the past year, we have designed and evaluated two RWV formats, based on the 50 ml STLV, which satisfy our gravity nullification requirements and are suitable for the construction of the SE. One modification (M1) involved machining four segmental compartments into the rear end cap of the STLV; the other modification (M2) has four identical size compartments machined into the vessel cylinder. In M1, DE, are cast simultaneously in the four compartments (generating segments of tissue), whereas in M2, DEs are cast in each compartment individually to produce rectangular pieces of tissue. Using either vessel, one RWV culture set up is used to generate four DE/SE under identical conditions. Since the experimental protocols involved in SE development require a minimum of 3 weeks, our modified RWVs also optimize hardware utilization. Dermal Equivalents Generated in RWV Fibroblast Phenotype: We have shown that the weight, volume, and therefore, density of DEs generated in 1g are independent of collagen type I concentration (3-5mg/ml) and cell number (fibroblasts, 105 - 5 x 105 cells/ml). Under these conditions, there is also no significant cell division or migration. This in vivo -like quiescence is maintained in the DE generated under microgravity during a 15-day observation period. Since fibroblasts respond to mechanical stimuli and are responsible for tissue contraction, developing DE is very sensitive to mechanical stresses. It was particularly important to observe that DEs generated in the RWV do not contract. In addition to indicating an absence of the "activated" fibroblast phenotype, the myofibroblast, this finding also suggests that the microgravity environment does not appear to impose significant mechanical stress on the DE. Treatment of the DE with neutral red confirmed cell viability. Paraffin-embedded, cross-sectioned DE showed a random distribution of cells throughout the matrix. In contrast, some "sedimentation" for fibroblasts is frequently observed under 1g conditions. Confocal microscopy: A more detailed and precise assessment of fibroblast distribution, orientation, and interaction with the collagen matrix will be obtained from studies of DEs by confocal microscopy (CFM). In order to facilitate such findings, we have developed methods for labeling fibroblasts with fluorescent Cell Tracker dyes, and incorporated the labeled viable cells into DEs. The utility of this technique was demonstrated by the analysis of orientation of a single fibroblast in a DE, which was defined by eight optical sections obtained at 9m intervals. Cell-matrix interactions are highly dependent on cell surface matrix receptors (integrins) and the extracellular components present. We are currently studying the expression of collagen-specific integrins and the organization of collagen fibers in DEs developed in the RWV. Mechanical Properties: Tensile strength of skin, primarily a feature of the dermis, is particularly important in the context of wound healing and clinical use of SEs as graft tissue. Preliminary experiments suggest that DEs generated in the RWV have a lower tangential stiffness (about 3-6 fold) than normal human dermis. Such differences in mechanical properties might be expected in view of the lower collagen fiber density in DE as shown by transmission electron microscopy. Metabolic Activity: Cell viability in DEs cultured in 1g can be maintained for the period of time required for epidermalization of the SE. Preliminary morphological data obtained from RWV experiments suggests that fibroblasts begin to show signs of distress after about 2 weeks. Initial metabolic studies suggest that glucose depletion or lactate accumulation are probably not contributing stress factors. Since the DE is submerged under a considerable volume of medium, hypoxic conditions might develop. Increasing oxygenation of the DE will be discussed below. Skin Equivalents Generated in RWV Construction: Although normal human keratinocytes and melanocytes are readily available in our laboratories, the initial focus is on the effect of microgravity on bicellular SE composed of fibroblasts and keratinocytes. Conditions for growth and differentiation of keratinocytes, which are also optimal for melanocyte survival, are being developed to facilitate the construction and studies of the tricellular SE. In a typical SE construction experiment, once DEs are established, the medium (Hams F12) is removed from the RWV, keratinocytes are placed on and allowed to attach to the surface of DEs (2-3 hours). The vessel is then charged with keratinocyte growth medium (KGM) and set to rotate. When the keratinocyte monolayer is confluent, differentiation is initiated by raising the extracellular calcium. Such developing SEs, when paraffin embedded and cross-sectioned, show that differentiation through several suprabasal layers (including the granular layer) does take place. The lack of cornification, however, suggests that for complete maturation, the SE needs to be cultured at an air-liquid interface (sufficient oxygen). This is consistent with our earlier work which showed that epidermalization of the SE is stimulated by exposure to hyperbaric oxygen. A stronger attachment of keratinocytes to the DE observed under RWV conditions indicates increased adhesion. This is manifested by increased tissue integrity at the dermal-epidermal junction observed during sectioning of embedded SEs. Expression of adhesion molecules by keratinocytes and melanocytes under microgravity is presently under study. SE Vessel: In order to provide conditions which will allow exposure of the SE surface to air and improve oxygenation and nutrition of the DE, we have developed a third format of the RWV. In this modification (M3), the vessel is composed of two parts, each of which has its own diffuser and medium exchange parts. the "vessel divider" is composed of two discs, each containing six wells, which sandwich a semipermeable membrane. The membrane forms the "floor" of each of the six wells, providing each well with an apical and a basal side. DEs are cast in the wells and the oxygenation and nutrients are supplied from both sides. Furthermore, SEs in the wells are at the air-liquid interface when one of the vessel changers is operated with very little or no medium. We are currently evaluating this vessel format, and preliminary experiments suggest that it will satisfy the required criteria. Confocal Microscopy of the SE: We believe that confocal microscopy will be a useful tool for studying processes involving cell-cell and cell-matrix interactions in the SEs as well as the DE. Consequently, we have shown that keratinocytes and melanocytes may be prelabeled with fluorescent Cell Tracker dyes. The fluorescence is retained in the cells through cell harvesting, seeding on DEs, proliferation, and differentiation. The SE labeled in this manner can be fixed, paraffin embedded, sectioned, and the cross-sections deparaffinized with retention of fluorescent label. Initially, this methodology will lead to the development of attachment and proliferation assays. Technology Transfer From the inception of this project, we have worked closely with Synthecon, Inc. Their experience in engineering and manufacturing of RWVs was instrumental in generating vessels suitable for our specific tissue assembly requirements. Prototypes of M1 and M2 were displayed at the Houston Investigators Working Group meeting earlier this year. An agreement is in place for the exclusive manufacture of the modified vessels, and these should eventually be available for general use.
Students Funded Under Research:				Task Initiation:	9/95	Expiration:	8/99
BS Students:	0	BS Degrees:	0	Project Identification:			962-23-01-37
MS Students:	1	MS Degrees:	0	NASA Contract No.:			NAG9-813
PhD Students:	1	PhD Degrees:	0	Responsible Center:			JSC