Efficacy of administered mesenchymal stem cells in the initiation and co-ordination of repair processes by resident disc cells in an ovine (Ovis aries) large destabilizing lesion model of experimental disc degeneration.

BACKGROUND: Forty percent of low back pain cases are due to intervertebral disc degeneration (IVDD), with mesenchymal stem cells (MSCs) a reported treatment. We utilized an ovine IVDD model and intradiscal heterologous MSCs to determine therapeutic efficacy at different stages of IVDD. METHODOLOGY: Three nonoperated control (NOC) sheep were used for MSC isolation. In 36 sheep, 6 × 20 mm annular lesions were made at three spinal levels using customized blades/scalpel handles, and IVDD was allowed to develop for 4 weeks in the Early (EA) and late Acute (LA) groups, or 12 weeks in the chronic (EST) group. Lesion IVDs received injections of 10 × 106 MSCs or PBS, and after 8 (EA), 22 (LA) or 14 (EST) weeks recuperation the sheep were sacrificed. Longitudinal lateral radiographs were used to determine disc heights. IVD glycosaminoglycan (GAG) and hydroxyproline contents were quantified using established methods. An Instron materials testing machine and customized jigs analyzed IVD (range of motion, neutral zone [NZ] and stiffness) in flexion/extension, lateral bending and axial rotation. qRTPCR gene profiles of key anabolic and catabolic matrix molecules were undertaken. Toluidine blue and hematoxylin and eosin stained IVD sections were histopathologically scoring by two blinded observers. RESULTS: IVDD significantly reduced disc heights. MSC treatment restored 95% to 100% of disc height, maximally improved NZ and stiffness in flexion/extension and lateral bending in the EST group, restoring GAG levels. With IVDD qRTPCR demonstrated elevated catabolic gene expression (MMP2/3/9/13, ADAMTS4/5) in the PBS IVDs and expession normalization in MSC-treated IVDs. Histopathology degeneracy scores were close to levels of NOC IVDs in MSC IVDs but IVDD developed in PBS injected IVDs. DISCUSSION: Administered MSCs produced recovery in degenerate IVDs, restored disc height, composition, biomechanical properties, down regulated MMPs and fibrosis, strongly supporting the efficacy of MSCs for disc repair.

Treatment of experimentally induced osteoarthritis in horses using an intravenous combination of sodium pentosan polysulfate, N-acetyl glucosamine, and sodium hyaluronan.

OBJECTIVE: To assess the effects of sodium pentosan polysulfate (PPS), N-acetyl glucosamine (NAG), and sodium hyaluronan (HA) in horses with induced osteoarthritis (OA). STUDY DESIGN: Experimental. ANIMALS: Adult Standard bred horses (n = 16). METHODS: OA was induced arthroscopically in 1 intercarpal joint; 8 horses were administered 3 mg/kg PPS, 4.8 mg/kg NAG, and 0.12 mg/kg HA (PGH), intravenously (IV), weekly and 8 horses were administered an equivalent volume of saline IV until study completion (day 70). Horses underwent a standardized treadmill exercise program. Clinical and radiographic findings and synovial fluid analysis were evaluated throughout the study. Macroscopic, histologic, histochemical, and biochemical findings were evaluated after necropsy. Comparisons of interest included OA and non-OA joints of saline treated horses and OA joints of PGH treated horses and OA joints of saline treated horses. Results were statistically analyzed with significance set at P < .05. RESULTS: OA caused increases in clinical assessment scores, synovial fluid variables, radiographic, macroscopic, and histologic cartilage scores, synovial fluid and cartilage chondroitin sulfate 846-epitope and glycosaminoglycan concentration. Total radiographic scores, total macroscopic joint pathology and macroscopic cartilage pathology scores were significantly reduced in horses treated with PGH compared with saline treated horses. Synovial fluid total protein concentration and white blood cell count were higher in OA joints of PGH treated horses compared with saline treated horses. There were no other significant differences between treatment groups. CONCLUSIONS: Improvements in macroscopic variables were not supported by other outcomes. Further evidence is needed before PGH can be recommended as a therapeutic option for osteoarthritis in horses.

Preliminary studies of alfaxalone for intravenous immobilization of juvenile captive estuarine crocodiles (Crocodylus porosus) and Australian freshwater crocodiles (Crocodylus johnstoni) at optimal and selected sub-optimal thermal zones.

OBJECTIVES: To investigate the character of immobilization given by alfaxalone in juvenile crocodiles at optimal and at suboptimal temperatures. STUDY DESIGN: Prospective, randomized partial crossover study. ANIMALS: Twenty captive male estuarine (weight 0.6-2.5 kg) and five captive male freshwater crocodiles (weight 0.2-0.6 kg). METHODS: Crocodiles were acclimatized for 24 hours at one of the following environmental temperatures; 32 °C, 27 °C, 22 °C or 17 °C, then received 3 mg kg(-1) intravenous (IV) alfaxalone into the dorsal occipital venous sinus. Duration and quality of immobilization was assessed and heart rate (HR) measured. On a separate occasion each crocodile was immobilized at one other environmental temperature. RESULTS: Alfaxalone, 3 mg kg(-1) IV, produced immobilization for 55 (range 15-100 minutes in estuarine, and 20 (range 20-25) minutes in freshwater crocodiles at 32 °C. There was no significant difference overall in immobilization times between temperatures, other than that, in estuarine crocodiles, duration was shorter at 32 °C than 22 °C. The character of immobilization was unpredictable, with animals recovering without warning, or having extended recoveries requiring assisted ventilation. Assisted ventilation was necessary mainly at the lower temperatures. Median HR in all temperature treatments decreased within 5 minutes post-injection, but the change in HR over the duration of immobilization was affected by the temperature, with a progressively smaller range of fall as temperature decreased. At 17 °C, two estuarine crocodiles appeared to re-immobilize after initial recovery, became severely bradycardiac and required ventilation and re-warming. CONCLUSIONS AND CLINICAL RELEVANCE: Alfaxalone IV in small captive estuarine and freshwater crocodiles provides adequate induction of immobilization at various temperatures. However, the unpredictable results following induction mean it is unsuitable for field use and should be restricted to environments where intubation and ventilation are available, where animals can be warmed to optimal temperature, and where access to immersion in water can be restricted for 24 hours.

The effect of short- and long-term treatment with manuka honey on second intention healing of contaminated and noncontaminated wounds on the distal aspect of the forelimbs in horses.

OBJECTIVES: To compare the effects of manuka honey and manuka honey gel on second intention healing of noncontaminated distal limb wounds and those contaminated with feces. STUDY DESIGN: Experimental study. ANIMALS: Standardbred horses (n = 10). METHODS: Five full-thickness wounds (2 × 2 cm) were created on both metacarpi. Wounds on 1 forelimb were covered with horse feces for 24 hours. Wounds on the contralateral limb were left uncontaminated. Wounds were assigned to the following 5 different treatments: manuka honey, manuka honey gel or gel applied for 12 days, manuka honey gel applied throughout healing and untreated control. Wound area was measured on day 1 then weekly until day 42 and time to complete healing was recorded. RESULTS: Wounds treated with manuka honey gel throughout healing healed faster than all other wounds (P < .05). Wounds treated with manuka honey and manuka honey gel for 12 days healed faster than gel control and untreated control wounds (P < .05). Wounds treated with manuka honey and manuka honey gel for 12 days and throughout healing were smaller than gel control and untreated control wounds until day 35 (P < .05). Wounds contaminated with feces had greater retraction for 7 days, but healed faster than noncontaminated wounds (P < .05). CONCLUSIONS: Treatment of wounds with manuka honey and manuka honey gel reduced wound retraction and overall healing time compared with gel and untreated control wounds.

A preliminary study on the effect of wounding on transforming growth factor-ß1 and cartilage oligomeric matrix protein concentrations in the skin of horses.

OBJECTIVE: To evaluate whether wound type or site influence the production of cartilage oligomeric matrix protein (COMP) and transforming growth factor ß1 (TGF-ß1), and determine if there is a correlation between TGF-ß1and COMP during healing. STUDY DESIGN: Experimental. ANIMALS: Standardbred horses (n=6), 4-8 years old. METHODS: Six, standardized, full-thickness skin wounds (open, straight, and elliptical) were surgically created on the neck (n=3) and metacarpus (3) on each horse. Wounds were randomly allocated to site and side. Tissue samples were collected before creating wounds and on days 7, 14, and 42. COMP concentration (µg/g dry weight of tissue) was determined using a standard competitive ELISA and TGF-ß1 (ng/g dry weight of tissue) was determined using a commercially available sandwich ELISA. RESULTS: COMP concentrations were higher in intact skin on the neck compared with the metacarpus (P=.02). There was no difference in COMP and TGF-ß1 concentrations between the different wound types or sites during healing. There was no correlation between TGF-ß1 and COMP during healing. CONCLUSIONS: Within the limitations of the study design, production of COMP during healing of skin wounds does not appear to be influenced by wound type or anatomic site, nor does it appear to be correlated with TGF-ß1 concentrations.

A preliminary study on the effect of manuka honey on second-intention healing of contaminated wounds on the distal aspect of the forelimbs of horses.

OBJECTIVE: To determine the effect of manuka honey on second-intention healing of contaminated, full-thickness skin wounds in horses. STUDY DESIGN: Experimental. ANIMALS: Adult Standardbred horses (n = 8). METHODS: One wound was created on the dorsomedial aspect of the third metacarpus in both forelimbs, contaminated with feces, and bandaged for 24 hours. Bandages were removed and wounds rinsed with isotonic saline solution. Wounds on 1 limb had manuka honey applied daily (n = 8) whereas wounds on the contralateral limb received no treatment (n = 8). Bandages were replaced and changed daily for 12 days, after which treatment stopped, bandages were removed, leaving wounds open to heal. Wound area was measured 24 hours after wound creation (day 1), then weekly for 8 weeks. Overall time for healing was recorded. Wound area and rate of healing of treated and control wounds were compared statistically. RESULTS: Treatment with manuka honey decreased wound retraction and treated wounds remained significantly smaller than control wounds until day 42; however, there was no difference in overall healing time between treatment and control wounds. CONCLUSIONS: Treatment with manuka honey reduced wound area by reducing retraction but did not affect overall healing time of full-thickness distal limb wounds using this wound-healing model.

Effect of prosthesis number and position on rima glottidis area in equine laryngeal specimens.

OBJECTIVE: To assess the effect of 1 or 2 laryngeal prosthetic sutures on rima glottidis areas in equine laryngeal specimens. STUDY DESIGN: Experimental, randomized design. ANIMALS: Cadaveric equine larynges (n=16). METHODS: Larynges were collected from 10 horses; 2 sutures each were preplaced in the right and left sides of each larynx. A dorsal suture (DS) was placed through the caudal rim of the dorsal midline of the cricoid cartilage, under the cricopharyngeus muscle and through the proximal and rostral aspect of the muscular process. A lateral suture (LS) was placed 1.5 cm lateral to the DS and through the muscular process more distal and caudal to the 1st suture. Larynges were positioned in a customized stand and the rima glottidis photographed after each suture (LS or DS) or suture combination (CS) was tied in random sequence. An additional 6 larynxes were used to determine whether the tension applied to the sutures was repeatable. Sutures were preplaced in both the right and left side of each larynx as described above and each suture and CS was tied and released 3 times in each larynx. Photographs were taken of the rima glottidis after each suture or CS was tied generating 3 replicates for each suture configuration on each side of the 6 larynges. RESULTS: Mean rima glottidis area was not different between DS and LS when tied alone (P=.85); however, mean area after CS (DS+LS) was greater than DS (P<.001) and LS (P<.001) alone. The coefficient of variation for the 6 suture patterns were low (1-7%) and the intraclass correlation coefficient estimates were very high (0.997-0.998) demonstrating excellent repeatability between replicates for each of the 3 suture configurations. CONCLUSION: Our results suggest that laryngoplasty using 2 prostheses; 1 placed dorsally in the cricoid and through the rostral and proximal muscular process and 1 placed 1.5 cm lateral to the 1st and more caudal and distal in the muscular process results in a greater cross sectional area of the rima glottidis than either suture used alone. CLINICAL RELEVANCE: Seemingly prosthetic sutures contribute independently to each other in determining the contour of the rima glottidis. Use of 2 prosthetic sutures improves crosssectional area of the rima glottidis compared with each suture alone and may improve surgical outcome in laryngoplasty.