Eight 8-mm circular skin biopsy samples were aseptically obtained from each of 3 healthy young horses using a Fray Biopunch® following local infiltration of lidocaine HCl. All animal experimentation was done with approval of MU ACUC. Skin samples were transferred into 25 ml HBSS (Hank’s Balanced Salt Solution) supplemented with 2% p/s/a (penicillin [10,000 U/ml]; streptomycin [10,000 µg/ml]; amphotericin B [250 µg/ml]) on ice. Skin samples were washed 8X with HBSS and 8X with DPBS (Dulbecco’s Phosphate Buffered Saline). Skin samples were then treated with 0.25% trypsin in HBSS + 2% p/s/a for 16 h at 4°C and a subsequent 30 minutes at 37°C in a 5% CO2 incubator. Five milliliters of DMEM-HG (Dulbecco’s Modified Eagle Medium – High Glucose) + 10% FBS (Fetal Bovine Serum) + 1% p/s/a were then added to the skin samples and allowed to stand for 3 minutes. Using a #10 scalpel blade, epidermis and dermis were separated. Epidermal tissue was re-suspended in DMEM-HG +10% FBS +1% p/s/a and debris was filtered by passage through a strainer. The re-suspended solution was centrifuged for 10 minutes at 1,000 rpm. The supernatant was discarded and residual epidermis was re-suspended with the DMEM-HG +10% FBS +1% p/s/a and plated onto 2 collagen-coated Flexplates™, which were transferred into a 37°C, 5% CO2, 90% humidity incubator until an 80% confluence of cell growth was achieved (Fig. C). Media were changed every 2-3 days. Once 80% confluency had been achieved, the Flexplates™ were assembled into the Flexercell® Tension PlusTM System (Flexcercell® Strain Unit, FlexCentral FX-4000) (Fig. E). Keratinocyte monolayers were subjected to 8% strain at 1.5 Hz for 12 h or 24 h (Fig. G). Control monolayers were cultivated simultaneously in the absence of applied strain (Fig. F).

Media were collected at the conclusion of each treatment period and loaded into 10% Zymogram Gelatin gels™ (Invitrogen) and electrophoresis was performed for 100 minutes. Each gel was then processed and stained using Simply Blue Safestain™ (Invitrogen). Immunohistochemical confirmation that confluent monolayers were comprised of keratinocytes was performed on residual cells using a murine-derived monoclonal pancytokeratin antibody (DakoCytomation) (Figs. H, I). Degradation of gelatin was manifested by zones of clearing on a blue-stained gel. Determination of apparent molecular weights of MMPs was made by reference to standard molecular weight markers.  Bands of lysis were examined using a computer software program (Image J™) in order to yield quantitative data for statistical analysis. One way ANOVA was performed for the 3 independent experiments to assess variability in MMP activity amongst experimental groups. Tukey’s test was utilized as a post hoc test and significance was set at p < 0.05.

Equine laminitis is characterized by separation of the secondary epidermal lamellae from secondary dermal lamellae. Increased matrix metalloproteinase (MMP) enzymes are pivotally responsible for dis-attachment of basal keratinocytes in the hoof lamellar interface from underlying lamellar basement membrane (LBM) (Figs. A, B).1 Both MMP-9 and MMP-2 are increased in homogenates of laminitic lamellar tissues compared with controls.2 In addition, expression of MMP-2 in laminitic tissues is elevated.3 In laminitis, PMN infiltration from the circulation likely contributes to increased MMP-9 activity, whereas, lamellar keratinocytes may contribute to the increased activity of both MMP-9 and MMP-2.3,4 Normal attachment of basal keratinocytes to the LBM depends on proper function of a junctional protein complex, the hemidesmosome (HD), along with anchoring fibrils (Figs. A, D).5 Although a moderate amount of active MMP is normally responsible for enzymatic remodeling of the epidermal lamellae, excessive production of active MMP at this location causes degradation of crucial structural components in the HD-LBM protein complex (Fig. B).6 Hoof is uniquely susceptible to any reduction in the strength of the keratinocyte-LBM because this interface is persistently under the influence of remarkable tensile force (bearing the weight of the horse).