Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment

Abstract

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

Development of Microfabricated Dermal Epidermal Regenerative Matrices to Evaluate the Role of Cellular Microenvironments on Epidermal Morphogenesis

“…29 A collagen-glycosaminoglycan (GAG) coprecipitate containing collagen (5 mg/mL) and GAG (0.18 mg/mL) was prepared. 30 The collagen-GAG coprecipitate was placed into an aluminum weigh boat, lyophilized, and crosslinked using thermal dehydration at 105°C in a vacuum of < 200 mtorr for 48 h. The collagen-GAG sponges were cut into rectangles approximately 7 cm 2 (2.5-cm width · 3-cm length) and placed in a desiccator until use.…”

“…30,32 Neonatal foreskins were obtained from nonidentifiable discarded tissues from the UMass Memorial Medical Center, Worcester, MA, and were approved with an exempt status from the New England Institutional Review Board. After 5 days of culture, NHKs were detached using 0.05% Trypsin-EDTA (Invitrogen), and passage 2-3 NHKs from multiple donors were used in all experiments.…”

Evaluation of a bilayered, micropatterned hydrogel dressing for full-thickness wound healing

“…5-mm and 50-mm spacings) ( Table 1) based on previously reported results, which demonstrated that channels of these sizes enhanced re-epithelialization and recapitulated the texture of the native epidermal-dermal junction. 17,36 Various aspect ratios (i.e. ratio of topography height to topography width) were tested to optimize micropattern height for guiding cell migration.…”

Combined Effects of Scaffold Stiffening and Mechanical Preconditioning Cycles on Construct Biomechanics, Gene Expression, and Tendon Repair Biomechanics

“…[10][11][12][13][30][31][32] If not applied appropriately, DHT crosslinking is well known to change the chemical composition of collagen-based membranes.…”

“…12,13,30 Any cytotoxicity of strongly crosslinked, collagen-based materials could modify cell shape and significantly reduce cell growth. 31 DHT crosslinking has also been postulated to significantly decrease the rate of cell migration by masking the integrin binding sites that promote cellular attachment.…”