Skin and hair on-a-chip: in vitro skin models versus ex vivo tissue maintenance with dynamic perfusion.

Substantial progress has been achieved over the last few decades in the development of skin equivalents to model the skin as an organ. However, their static culture still limits the emulation of essential physiological properties crucial for toxicity testing and compound screening. Here, we describe a dynamically perfused chip-based bioreactor platform capable of applying variable mechanical shear stress and extending culture periods. This leads to improvements of culture conditions for integrated in vitro skin models, ex vivo skin organ cultures and biopsies of single hair follicular units.

A dynamic multi-organ-chip for long-term cultivation and substance testing proven by 3D human liver and skin tissue co-culture.

Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, to accurately predict drug toxicity. In this study, we present a multi-organ-chip capable of maintaining 3D tissues derived from cell lines, primary cells and biopsies of various human organs. We designed a multi-organ-chip with co-cultures of human artificial liver microtissues and skin biopsies, each a (1)/100,000 of the biomass of their original human organ counterparts, and have successfully proven its long-term performance. The system supports two different culture modes: i) tissue exposed to the fluid flow, or ii) tissue shielded from the underlying fluid flow by standard Transwell® cultures. Crosstalk between the two tissues was observed in 14-day co-cultures exposed to fluid flow. Applying the same culture mode, liver microtissues showed sensitivity at different molecular levels to the toxic substance troglitazone during a 6-day exposure. Finally, an astonishingly stable long-term performance of the Transwell®-based co-cultures could be observed over a 28-day period. This mode facilitates exposure of skin at the air-liquid interface. Thus, we provide here a potential new tool for systemic substance testing.

De novo formation and ultra-structural characterization of a fiber-producing human hair follicle equivalent in vitro.

Across many tissues and organs, the ability to create an organoid, the smallest functional unit of an organ, in vitro is the key both to tissue engineering and preclinical testing regimes. The hair follicle is an organoid that has been much studied based on its ability to grow quickly and to regenerate after trauma. But hair follicle formation in vitro has been elusive. Replacing hair lost due to pattern baldness or more severe alopecia, including that induced by chemotherapy, remains a significant unmet medical need. By carefully analyzing and recapitulating the growth conditions of hair follicle formation, we recreated human hair follicles in tissue culture that were capable of producing hair. Our microfollicles contained all relevant cell types and their structure and orientation resembled in some ways excised hair follicle specimens from human skin. This finding offers a new window onto hair follicle development. Having a robust culture system for hair follicles is an important step towards improved hair regeneration as well as to an understanding of how marketed drugs or drug candidates, including cancer chemotherapy, will affect this important organ.

Design and prototyping of a chip-based multi-micro-organoid culture system for substance testing, predictive to human (substance) exposure.

Dynamic miniaturized human multi-micro-organ bioreactor systems are envisaged as a possible solution for the embarrassing gap of predictive substance testing prior to human exposure. A rational approach was applied to simulate and design dynamic long-term cultures of the smallest possible functional human organ units, human "micro-organoids", on a chip the shape of a microscope slide. Each chip contains six identical dynamic micro-bioreactors with three different micro-organoid culture segments each, a feed supply and waste reservoirs. A liver, a brain cortex and a bone marrow micro-organoid segment were designed into each bioreactor. This design was translated into a multi-layer chip prototype and a routine manufacturing procedure was established. The first series of microscopable, chemically resistant and sterilizable chip prototypes was tested for matrix compatibility and primary cell culture suitability. Sterility and long-term human cell survival could be shown. Optimizing the applied design approach and prototyping tools resulted in a time period of only 3 months for a single design and prototyping cycle. This rapid prototyping scheme now allows for fast adjustment or redesign of inaccurate architectures. The designed chip platform is thus ready to be evaluated for the establishment and maintenance of the human liver, brain cortex and bone marrow micro-organoids in a systemic microenvironment.

Submural histopathologic changes attributable to peracute laminitis in horses.

OBJECTIVE: To describe submural histopathologic changes attributable to peracute laminitis in horses. ANIMALS: 20 adult horses. PROCEDURE: A concurrent-control design was used to compare laminar lesions in 10 horses subjected to carbohydrate-induced laminitis with laminar characteristics of 10 sex- and aged-matched control horses with normal feet. Horses in the treatment group were administered an overload of carbohydrate. Tissues were obtained by biopsy 4 to 8 hours after onset of lameness or 72 hours after administration of the carbohydrate overload when lameness did not develop. Sections were stained with H&E, Masson's trichrome, and periodic acid-Schiff stains. Histopathologic changes were analyzed to detect differences between groups and to correlate epidermal changes with severity and duration of lameness. RESULTS: Analysis indicated that dermal and epidermal lesions were evident despite lack of visible separation of the epidermal basement membrane, can be found in horses without detectable lameness, and were nonspecific and progressive following onset of lameness. Furthermore, severity and location of lesions were associated with severity and duration of lameness. CONCLUSION AND CLINICAL RELEVANCE: These observations are consistent with the concept that separation of the laminar epithelial basement membrane is a delayed step in the pathogenesis of acute laminitis, digital vascular hypoperfusion is an underlying cause for laminitis, and the potential for repeated episodes of subclinical laminitis may underlie the development of structural and mechanical changes consistent with chronic laminitis despite lack of clinical signs of acute laminitis.

Evaluation of systemic immunologic hyperreactivity after intradermal testing in horses with chronic laminitis.

OBJECTIVE: To determine whether systemic immunologic hyperreactivity exists in horses with chronic laminitis, compared with responses for nonlaminitic horses. ANIMALS: 7 nonlaminitic horses and 7 CL horses. PROCEDURE: In experiment 1, intradermal testing (IDT) was performed on 7 nonlaminitic and 7 CL horses to evaluate the response to a combination of 70 allergens at 15 and 30 minutes and 4 and 24 hours after injection. Three nonlaminitic and 3 CL horses used in experiment 1 were used in experiment 2 to determine whether histologic differences existed between the 2 groups. The H&E-stained tissue sections were evaluated on the basis of 3 criteria. For all analyses, 2-sample t-tests were used to determine significant differences between the groups. RESULTS: In experiment 1, CL horses had significantly higher total responses to IDT than nonlaminitic horses at the first 3 time periods. Also, CL horses had significantly fewer total scores of 0 than nonlaminitic horses at all time periods, except at 24 hours. In experiment 2, we did not detect significant differences between groups for any criterion. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the hypothesis that CL horses develop hyperreactivity to various antigenic stimuli, compared with responses for nonlaminitic horses. Therefore, the possibility that antigenic challenge may result in exacerbation of clinical signs of laminitis should be discussed with horse owners. Chronic laminitis should also be a consideration when a horse becomes lame following antigenic challenges.

Short-term effect of therapeutic shoeing on severity of lameness in horses with chronic laminitis.

OBJECTIVE: To evaluate the short-term effects of 4 therapeutic shoeing systems on lameness and voluntary limb-load distribution in horses with chronic laminitis. ANIMALS: 10 horses with chronic laminitis. PROCEDURES: A clinical trial was conducted that used a concurrent control, crossover design to evaluate the relative effectiveness of a standard flat shoe, fullered egg-bar shoe, heart-bar shoe, and modified equine digital support system to alleviate chronic lameness in horses. Therapeutic success was assessed during a 7-day period by use of subjective (Obel grade and clinical score) and objective (force-plate data) evaluations. RESULTS: Comparison of pretreatment and intertreatment control data indicated that disease status of the horses did not change during the course of the study. None of the therapeutic shoeing treatments used resulted in a significant change in severity of lameness. CONCLUSIONS AND CLINICAL RELEVANCE: Results were interpreted to imply that substantial clinical improvement should not be expected during the first 7 days after therapeutic shoeing for the specific shoes tested in this study. On the basis of our results, we hypothesize that when used as the lone indicator of therapeutic success, severity of lameness may not be a valid indicator.

Effect of prolonged water immersion on equine hoof epidermis in vitro.

OBJECTIVE: To evaluate the effect of prolonged water exposure on tissue mass and solutes of outer and inner layers of the stratum medium, sole, frog, and the stratum medium (SMZA) zona alba layer of horses' hooves. SPECIMEN POPULATION: 10 hooves from 10 horses without foot abnormalities. PROCEDURES: Hoof wall tissue specimens were obtained and immersed for 10 days in distilled deionized water. Serial changes in mass were recorded during the immersion period. Subsequently, osmolarity and Na+, K, Cl-, and protein concentrations of the immersion solution were quantified. RESULTS: Fully cornified outer hoof wall, sole, and frog epidermal structures increased in mass, whereas the SMZA lost mass when immersed in water. All hoof structures had a variable loss of crystalloids during immersion, but none of the specimens lost proteins. The frog epidermis was distinct in that total solute lost during immersion could not be ascribed to N+, K+, and Cl-. CONCLUSIONS AND CLINICAL RELEVANCE: Data support a 2-compartment model for the fully cornified outer stratum medium, frog, and sole that permits the exchange of crystalloids, but not proteins, across the cell membrane and infers that topical agents containing proteins cannot benefit the hoof. The unique osmotic behavior of the SMZA relative to other hoof structures suggests the hypothesis that it is composed of transitional epithelial cells. The solutes lost from frog epithelium are interpreted to reflect its unique lipid composition.

Effectiveness of glyceryl trinitrate for enhancing digital submural perfusion in horses.

OBJECTIVE: To evaluate the clinical efficacy of topically administered glyceryl trinitrate (GTN) for inducing digital submural vasodilation in clinically normal horses. ANIMALS: 7 adult horses without foot abnormalities. PROCEDURES: A concurrent-control crossover design was used to determine whether topical application of GTN ointment for prevention or treatment of laminitis would result in a detectable increase in digital perfusion. Heat-acclimated horses instumented for detection of wall surface temperature (HWST), mean systemic pressure, and heart rate were used. Horses were exposed to cold to induce digital vasoconstriction and treated with GTN in an attempt to induce digital vasodilation. RESULTS: Application of GTN failed to induce an increase in digital submural perfusion but did induce a mild decrease in mean systemic pressure. CONCLUSIONS AND CLINICAL RELEVANCE: Topical application of 60 mg of GTN as a 2% ointment on the skin over the major vasculature in the region of the proximal interphalangeal joint (pastern) of horses was not effective in significantly increasing digital perfusion. A decrease in mean systemic pressure following treatment was observed, implying that the drug was absorbed. Use of GTN may result in a decrease in digital submural perfusion secondary to induction of peripheral constriction or a decrease in digital perfusion pressure.

Effectiveness of a unique dihydropyridine (BAYTG 1000) for prevention of laminitis in horses.

OBJECTIVE: To determine whether a unique dihydropyridine (BAYTG 1000) would be beneficial in preventing laminitis in horses. ANIMALS: 16 clinically normal adult horses. PROCEDURE: 8 pairs of horses were used in a controlled double-blind study, using sex- and age-matched horses randomly assigned to treatment or control groups. Horses were subjected to carbohydrate overload to induce laminitis. Treated horses were administered BAY TG 1000 (30 mg/kg, PO, q 24 h) for 3 days. Hoof wall surface temperature (HWST) and lameness were recorded at 4-hour intervals. The HWST was adjusted on the basis of time of onset of lameness and evaluated, using a repeated-measures ANOVA. Lameness 8 hours after onset and clinical status 72 hours after onset of lameness were evaluated, using Mann-Whitney procedures. RESULTS: Analysis revealed that BAYTG 1000 did not decrease the incidence of lameness but significantly ameliorated prodromal hypothermia, lessened the severity of lameness 8 hours after onset of lameness, and improved the clinical status of horses 72 hours after onset of lameness. CONCLUSION AND CLINICAL RELEVANCE: Results support the conclusion that BAYTG 1000 was protective when used in prevention of laminitis. The drug decreased severity and improved clinical status (recovery) of induced lameness, which was interpreted to mean that the drug's actions were on mechanisms important but secondary to primary causal mechanisms of laminitis. Therefore, drugs that enhance digital perfusion via alteration of rheologic activity may have potential use in the prevention and management of laminitis in horses.