Animals

Female (n= 5) and male (n= 5) horses of different breeds, age and different activities afflicted by chronic laminitis were used to conduct this study (Table 1), none of the horses were under any current treatment. All of the animals displayed distal phalanx rotation but were not sinkers. Blood cell count (CBC) was drawn from the jugular vein was used to appraise the initial inflammatory state. All horses were kept and treated in their corresponding regular stalls. Animals had free access to water and were fed with oat hay twice a day. Informed consent forms were signed by the owners. Horse clinical and experimental procedural and handling protocols were approved by the Veterinary College (FMVZ-UNAM) Institutional Animal Care and Use of Animal Committee (No. MC-2016/2-5; “Evaluación terapéutica del uso de células troncales mesenquimales alogénicas, en el control del dolor, inflamación y estructura lamelar en caballos con lamintis crónica”).

Lameness assessment

An orthopedic evaluation was made by observing the static and dynamic postures of each horse. It was used the modified multifactorial evaluation pain score adapted for laminitic horses that rates signs of pain and altered behavior by combining, respectively, Obel’s and Glasgow’s criteria⁴. The highest the score (maximum 14), the greatest the pain.

Hooves temperature assessment

Lamellar inflammation leads to increments in hooves temperature²⁰. Infrared thermography may be used to estimate the change in temperature through hoof imaging. It was utilized the infrared FLIR XX5 camera (Flir^TM, USA) to image real time front limb

hooves of horses once a month for six months. The temperature of the coronary band was recorded in situ and graphed individually. It was decided to use the coronary band as an anatomical reference to measure the temperature because it lacks hoof wall. This feature makes this region amenable for obtaining reliable temperature readings; the anatomy of hoof wall in chronic laminitic horses may become greatly deformed. Imaging sessions were conducted at the same hour (( 8 AM), while keeping horses stood in its stall and before being hand-walked. Hooves were always cleaned before imaging. Lateral and dorsal views were taken at a distance of 30 centimeters away from the hoof. Color coded images were obtained based on a calibrated linear scale built up on the camera acquisition program.

Venography

Horses were sedated with 10% xylazine hydrochloride injected through the jugular vein. The lateral and medial palmar digital nerve were blocked using 2% lidocaine at level of proximal sesamoids; previously the area was shaved and cleaned with a solution containing 20% chlorhexidine gluconate. A tourniquet was placed with an Esmarch bandage at the fetlock. The lateral digital vein was then punctured using a butterfly catheter 23G and the contrast medium Iopamidol (Scanlux, San Chemia, México) injected through it slowly. To assure an adequate distribution of Iopamidol throughout the entire horse’s digit vascular bed, the knee dorsal aspect was flexed dorsally, and the heels lifted from the ground. At the end of the administration, the heels were weight bearing, the syringe removed, and the catheter clamped with a Halsted tweezers. Latero-medial and dorso-palmar views were taken at 60 sec from the contrast medium²¹.

Lamellar biopsy

The fetlock and pastern were washed and dried. Horses were sedated and abaxial lateral and medial nerves blocked following the guidelines provided above. A tourniquet was placed in the fetlock using an Esmarch bandage to reduce bleeding. The dorsal hoof wall was drilled with a 4.8 mm diameter polishing stone (Dremel, Bosch, USA), 2 cm distal from the coronary band. The cavity made (9 mm diameter) traversed the external and medium strata of the hoof’s wall until reaching the white line, an anatomical landmark that indicates proximity to the lamellar corium. Saline solution was constantly irrigated to avoid overheating during drilling. Once in the white line, the laminar corium was incised with a scalpel armed with a No. 11 blade, oriented perpendicular to the hoof wall. The scalpel was moved clockwise following the perimeter of the cavity until reaching the third phalanx. Then, with a No. 4 Frahm’s scaler, the laminar tissue was removed from third phalanx²². A cylindrical-shaped laminar sample, measuring approximately 7 mm at the base and 7 mm in height, was obtained. The biopsy was fixed in buffered paraformaldehyde (4%) for 24 h, then submerged into a buffered sucrose solution (15 %) for 24 h, and finally transferred to a buffered 30% sucrose solution at 4 (C until evaluation. The surgical area was dried with gauzes and sealed with methyl-methacrylate. The tourniquet was removed after methyl-methacrylate dried.

Histology

Biopsies were embedded in Tissue-Tek (Neg-50(, Richard-Allan Scientific, USA), frozen in dry ice and cut longitudinally (30 μm thick) in a cryostat (SLEE), at -25 °C. Serial sections were mounted on to gelatin subbed slides and stored at -4 °C until used. A total of 15 slides were obtained per sample. A slide was taken every third to be stained with Cresyl violet, Periodic Acid-Schiff’s base and Masson’s Trichromic stain. Slides were cover slipped with Cytoseal^TM 60 (Richard-Allan Scientific) and observed through an Olympus BX51-WI microscope. The tissue cytological integrity and degree of inflammatory infiltration was estimated qualitatively, and representative fields of this material were digitally photomicrographed, under bright-field microscopy, by using the Stereo Investigator( Software (10X).

Transmission electronic microscopy

Samples of the lamellar corium obtained from a healthy and a laminitic horses were fixed in a tampon solution containing 4% paraformaldehyde / 2.5% glutaraldehyde (Electron Microscopy Sciences) for 72 h. After three gentle washes with phosphate buffer-saline (0.1M; pH 7.2), the samples were post-fixed with osmium tetroxide (1%) for 1 h, then dehydrated and embedded in Epon 812. Polymerized at 60 (C for 24 h. Semi-thin sections (150 nm) were mounted in slides and dyed with Toluidine blue, the interest area was chosen and Ultra-thin sections (70 nm) were mounted on cupper grids, contrasted with uranyl acetate (2%) and lead citrate (50%) (Electron Microscopy Sciences) and observed through a Jeol 1010 transmission electron microscope (60kV). The tissue ultrastructural integrity and degree of inflammatory infiltration was estimated qualitatively, and representative fields of this material were digitally photomicrographed.

Bone marrow sampling and mesenchymal stem cell collection, isolation and expansion

Bone marrow (BM) samples (15 mL) were collected (using 20 mL syringes containing 1,000 UI heparin/ml BM) through a sternal puncture from male and female healthy horses (3-8 yr-old) of different breeds. Samples were stored at 4 - 8 (C and transferred in aseptic conditions to the cell culture facility within an hour from collection. Bone marrow was transferred to sterile tubes filled with Ficoll Hypaque at a 1:2 proportion. The samples were centrifuged at 400 xg for 30 min and the mononuclear fraction was isolated in a sterile tube at 4(C. The fraction was centrifuged at 300 xg for 10 minutes at 4 (C. The supernatant was discarded and the cells were re-suspended and seeded, at a density of 5x10⁴ cells/cm², in 25 cm³ flasks containing DMEM:F12 (1:1) supplemented with 20% fetal bovine Serum, 500 U/mL of penicillin, 50 (g/mL of streptomycin and 2.5 (g/mL amphotericin B. The cells were incubated at 37 °C and 5% CO₂ and the culture media were changed every three days until reaching an 80% confluence²³.

Equine mesenchymal stem cell subculture and scaling

After the 80 % confluence was reached, the cells were sub-cultured weekly as follows: the monolayer was washed with DPBS and the cells were detached using HBSS with 7.5 mg of porcine trypsin and 0.6 mg of EDTA. The cells were seeded at a density of 5x10⁴ cells/cm² in 75 cm³ flasks (first subculture) and then in 175 cm³ (subsequent subcultures) with DMEM F12 (1:1) supplemented with 20% Fetal Bovine Serum.

Flow-cytometry

Allogenic, bone marrow mesenchymal stem cells were characterized through flow-cytometry. Three sets of antibodies were used to immunophenotype them. ABM-MSC used for infusion were positive to the surface markers CD73, CD90, CD105, CD28 and CD44 (CD28/CD44 specific for equine MSC), negative to CD45, CD34, CD14 and CD79. Flow cytometry was conducted based upon protocols previously reported^24-28.

Administration of ABM-MSC’s

Horses were sedated and prepared for administration of ABM-MSC’s as previously described (see venography section). ABM-MSCs were diluted in physiologic saline solution and administered (10-30 x 10⁶) into the lateral (or medial) digital palmar vein under sterile conditions via a 21-gauge butterfly catheter, attached to a 20 mL syringe, in the standing horse. Tourniquet was removed 20 min after administration of cells. The digit was kept bandaged for one day. ABM-MSCs were injected three times at one-month intervals.

Clinical condition of horses treated with ABM-MSCs

Horses with chronic laminitis had Obel-Glasgow’s scores that range between 2 to 14 before ABM-MSCs treatment onset (Figure 1A). In contrast, the healthy horse displayed neither lameness or pain, nor hooves temperature increments at any time along the period evaluated. As the study progressed, all ABM-MSCs treated horses improved their clinical condition after the second or third month of having received ABM-MSCs venous infusions. By the end of the six months’ period, all ABM-MSCs treated horses decreased their Obel-Glasgow scores and significantly improved their mobility and clinical condition. Overall, the horses with the worst initial condition, improved the most (Figure 1A). All horses had an improvement in mobility, quality and shape of the hoof, they also improved their behavior and the factions of their faces (Grimace scale), having a good quality of life. This improvement was attributable to ABM-MSCs infusion, since the sham horse treated only with vehicle did not improve through the course of the experimental time (Figure 1A).

(A) ABM-MSCs treated horses, monitored by the Obel-Glasgow scale and infrared thermography, (B) Hooves temperature.

Figure 1 Clinical evolution of horses after being treated with allogenic bone marrow, mesenchymal stem cells (ABM-MSCs) 

In contrast, hooves’ temperature in laminitic horses ranged between 27 (C and 35.5 (C (average 33.26 (C + 2.58) before ABM-MSCs treatment started. As a reference, the temperature in the healthy horse’s hooves ranged between 27 (C and 30 (C (average 28.56 (C + 0.79). ABM-MSCs treatment had no major impact on hooves temperature (Figure 1B). In a handful of treated horses, hoof’s temperature tended to decrease slightly (1 to 3 (C) after ABM-MSCs infusions (average 32.27 (C + 1.78). In others, temperature shifts were highly variable throughout the experimental timeline. In all cases, nonetheless, ABM-MSCs treated horses and the sham horse (average 33.01 (C + 1.29) never reached steady healthy values of hooves’ temperature.

Hooves vascular pattern and histology of the laminar corium after ABM-MSCs treatment

The horse’s hooves are irrigated by a highly anastomosed vascular bed formed by the coronary plexus and papillae, the sublamellar venous plexus, the terminal papillae, the circumflex vessels and the sole and heel’s venous plexuses (Figure 2 ). In laminitic horses, the chronic dislocation stands of the distal phalanx significantly disrupted this neat vascular pattern. As seen in Figure 2B, hooves virtually become avascular; the entire vascular bed showed severe contrast media filling defects except for stem vessels in coronary and heel’s plexus where vessels looked irregular and somewhat engorged. In general, the severity of vascular damage correlated with the severity of the symptoms (not shown). In contrast to the venographic findings described for the vascular landscape in laminitic hooves, images from the hooves obtained by the end of the 6^th month of having begun ABM-MSC treatment revealed, in all horses regardless of the initial clinical condition and degree of vascular damage, hints of vascular recovery. Commonly, stem vessels of the coronary and heel plexus became widened and images of vascular sprouts were frequently observed as were vessels traversing somewhat “aberrant” trajectories (Figure 2C). In other words, a process of angiogenesis is clearly on its way. None of the horses treated, however, showed a full recovery of the vascular pattern, even though their clinical improvement was evident. Lastly, the sham horse showed no clinical improvement, nor venographic patterns of vascular recovery (not shown).

A-C. Representative hoof venographies of a healthy (A), laminitic (B) and ABM-MSCs infused horses (C). Notice the vascular response in hooves of ABM-MSCs-infused horses. CP, Coronary Plexus; SP: Sublamellar Plexus; DPA: Distal Phalanx Apex; TP: Terminal Plexus; CV: Circumflex Vessels; A: Terminal Arch of the palmar digital vessels; B: Heel plexus; SVP: Soleal Venous Plexus. D-L. Representative photomicrographs of sagittal sections of the laminar corium of a healthy (D, G, J), laminitic (E, H, K) and ABM-MSCs infused horses (F, I, L) stained with Cresyl Violet (cell nuclei; D-F), PAS (Basal membrane; G-I) or Masson’s (Collagen; J-L) histochemical techniques. Notice the restoration of the laminar corium in ABM-MSCs infused horses. PEL: Primary Epidermal Lamellae; PDL: Primary Dermal Lamellae; SEL: Secondary Epidermal Lamellae; SDL: Secondary Dermal Lamellae; BM: Basal Membrane. M-O. Representative electron micrographs showing ultrastructural features of basal cells in healthy (M), laminitic (N) and ABM-MSCs infused horses (O). Notice the recovery of the chromatin and tonofilaments arrangement, as well as of desmosomes in ABM-MSCs infused horses. Nu: Nucleus; Tf: Tonofilaments; Ds and arrowheads: Desmosomes; (: oedema; Arrows in insets pinpoint the basal membrane. Refer to the text for a detailed description of Figure 2.

Figure 2 Vascular pattern and histology of the laminar corium after ABM-MSCs treatment  

Histological features

ABM-MSC-infused, but not the sham, horses improved their clinical condition and their hooves’ vascular patterns by the end of the 6^th mo of treatment. To evaluate whether this recovery might be associated to hooves’ tissue restoration, it was biopsied and histologically evaluated the laminar corium of a healthy, sham/laminitic and laminitic horses; the second and the third groups were sampled before and by the end of the treatment, 6 mo later from the first biopsy taken. Representative results are exhibited in Figure 2. In the healthy horse’s hoof (Figure 2D), it was observed primary epidermal and dermal lamellae interdigitating regularly. Secondary epidermal and dermal lamellae, on the other hand, ran in opposite directions, but parallel next to each other. Secondary lamellae displayed rounded ends thanks to the basal cell’s covering that progressed continuously up to the tip. The epidermal basal cells’ nuclei were juxtaposed opposite to the basal membrane (not shown). Finally, the basal membrane formed a thin and continuous layer underneath the epithelia lining (Figure 2G) and the collagen regularly organized in parallel bundles across the dermis (Figure 2J). In sharp contrast, the hooves histology in horses with chronic laminitis showed a virtual loss of the laminar architecture (Figure 2E), basal cell hyperplasia with numerous picnotic cell profiles, (Figure 2H), abundant connective tissue, as well as perivascular mononuclear cell infiltration (not shown). No basal membrane (Figure 2H) or collagen (Figure 2K) was observed. Finally, ABM-MSC-infused, but not the sham, horses’ hooves regained the laminar arrangement, the integrity of the epithelial monolayer upon a regular, continuous basal membrane and the organization and presence of collagen (Figure 2F, I and L). No evidence of significant perivascular mononuclear infiltration was detected.

Lastly, it was conducted electron microscopic observations to further evaluate the epithelial integrity in hooves of ABM-MSC-infused horses. In the healthy horse (Figure 2M), basal cells join one another by numerous desmosomes. Their eu-chromatic nuclei displayed irregular shapes characterized by indentations. They also presented a conspicuous cytoplasmic arrangement of tonofilaments. The electron dense basal lamina they were attached on was thin, regular and continuous. Laminitic hooves (Figure 2N), on the other hand, showed scarce desmosomes, abundant electro-lucid intercellular spaces. Basal cells tended to show nuclei with chromatin clumps. Cytoplasmic tono-filament aggregates were also frequent, and no evidence of basal membrane was observed. Hooves in ABM-MSC-infused horses (Figure 2O) showed a marked reduction of electro-lucid intercellular spaces. Desmosomes become a regular finding and cell nuclei return to their regular shape and chromatin arrangement.