Castration delays epigenetic aging and feminizes DNA methylation at androgen-regulated loci.

In mammals, females generally live longer than males. Nevertheless, the mechanisms underpinning sex-dependent longevity are currently unclear. Epigenetic clocks are powerful biological biomarkers capable of precisely estimating chronological age and identifying novel factors influencing the aging rate using only DNA methylation data. In this study, we developed the first epigenetic clock for domesticated sheep (Ovis aries), which can predict chronological age with a median absolute error of 5.1 months. We have discovered that castrated male sheep have a decelerated aging rate compared to intact males, mediated at least in part by the removal of androgens. Furthermore, we identified several androgen-sensitive CpG dinucleotides that become progressively hypomethylated with age in intact males, but remain stable in castrated males and females. Comparable sex-specific methylation differences in MKLN1 also exist in bat skin and a range of mouse tissues that have high androgen receptor expression, indicating that it may drive androgen-dependent hypomethylation in divergent mammalian species. In characterizing these sites, we identify biologically plausible mechanisms explaining how androgens drive male-accelerated aging.

Mastectomy in 25 small ruminants (2002-2019).

OBJECTIVE: To describe the outcome of small ruminants treated with unilateral and bilateral mastectomy by using three surgical techniques. STUDY DESIGN: Retrospective study. ANIMALS: Twenty-five small ruminants (24 goats and one sheep). METHODS: Medical records of animals that underwent mastectomy between November 1, 2002, and May 1, 2019, were reviewed. Follow-up information was obtained by telephone questionnaire with owners. Signalment, surgical data, intraoperative and postoperative complications, bacterial culture results, histopathologic diagnoses, short- and long-term outcomes, and other procedures performed were recorded. RESULT: Procedures consisted of six unilateral (with an elliptical incision) and 19 total (with inverted cloverleaf or elliptical skin incisions) mastectomies. All animals survived to hospital discharge. Intraoperative complications included contamination of the surgical site with mammary-gland fluid, hemorrhage, and difficulty dissecting skin from the mammary gland. Postoperative complications included seroma formation (7/25), surgical-site infection (5/25), and dehiscence of the skin incision (3/25). Mammary neoplasia was diagnosed in seven of 15 animals with histopathologic examination. No association was detected between surgical technique, diagnosis of neoplasia, and long-term outcome. Overall, client satisfaction was high. CONCLUSION: Mastectomy was effective at removing abnormally enlarged udders secondary to chronic mastitis, inappropriate lactation, idiopathic causes, or neoplasia and was associated with a low rate of complications in small ruminants. CLINICAL SIGNIFICANCE: Unilateral mastectomy with an elliptical skin incision or total mastectomy, preferably with inverted cloverleaf skin incision, may be indicated to remove diseased mammary tissue in small ruminants and can result in long-term survival with low morbidity and cosmetically pleasing results.

Risk factors for, frequency, and type of complications after temporary tube cystostomy in goats, sheep, and pigs.

OBJECTIVE: To describe risk factors for as well as type and frequency of complications, time to occurrence of complications, and mortality after temporary tube cystostomy alone or in combination with other surgical procedures, in goats, sheep, and pigs. STUDY DESIGN: Retrospective case series. ANIMALS: One hundred thirteen goats, eight sheep, and six pigs. METHODS: Medical records of goats, sheep, and pigs that underwent temporary tube cystostomy alone or in combination with urethrotomy at the Veterinary Medicine Teaching Hospital, University of California-Davis from January 1, 2007 to December 31, 2018 were reviewed. Signalment, presurgical and surgical procedures, urolith type and location, and postoperative complications and mortality were analyzed. In goats, logistic regression analysis was performed to determine association between the presence of complications or mortality as well as candidate predictor variables. RESULTS: Postoperative complications were reported in 54.9% (62/113) of goats, 37.5% (3/8) of sheep, and 50% (3/6) of pigs. The most frequent type of complication was reobstruction. Median (range) time to first complication was 171 days (1-2247) for goats. In sheep and pigs, the range for time to re-presentation for complications was 11 to 37 and 17 to 1182 days, respectively. Proportions of nonsurvivors after tube cystostomy were 48.4% (30/62) goats, 1/8 sheep and 2/6 pigs. No association between examined predictor variables and the occurrence of complications or mortality was detected. CONCLUSION: The risk of complications and mortality after temporary tube cystostomy was high, especially in goats. CLINICAL SIGNIFICANCE: Owners should be made aware of the high complication rate after temporary tube cystostomy alone or in combination with other procedures. Investigation of alternative approaches to manage obstructive urolithiasis in goats, sheep, and pigs should be considered.

Influence of two rumen cannulation techniques on postoperative recovery in sheep.

OBJECTIVE: To determine the influence of two methods of rumen cannulation on postoperative animal welfare. STUDY DESIGN: Experimental study. ANIMALS: Twelve commercial wethers (n = 6 per group). METHODS: Group 1 wethers underwent a one-step procedure to place a cannula immediately after fistulation of the rumen to the skin. Group 2 wethers underwent a two-step procedure in which a portion of the rumen was externalized and held with a clamp for 9 days, after which the cannula was placed into the fistula created by removal of the clamped rumen tissue. Feed intake and vital signs were monitored daily for 24 days postoperatively. Plasma fibrinogen and serum cortisol were measured daily for 14 days postoperatively to estimate inflammatory and stress responses, respectively. Change in body weight was also assessed. RESULTS: Cannulation method did not affect (P > .05) body weight, temperature, heart rate, respiratory rate, fibrinogen level, or cumulative cortisol level. Feed intake was lower (0.82 vs 1.2 kg/d; P < .0001), and mean cortisol level was greater (124.2 vs 121.5 ng/mL; P = .038) in group 2 compared with group 1. CONCLUSION: Although both cannulation methods mostly elicited similar physiological responses, animals seemed to experience more discomfort and stress when undergoing the two-step procedure. CLINICAL SIGNIFICANCE: Rumen cannulation performed in one step is recommended to improve postoperative welfare.

Validation of the Unesp-Botucatu composite scale to assess acute postoperative abdominal pain in sheep (USAPS).

A scale with robust statistical validation is essential to diagnose pain and improve decision making for analgesia. This blind, randomised, prospective and opportunist study aimed to develop an ethogram to evaluate behaviour and validate a scale to assess acute ovine postoperative pain. Elective laparoscopy was performed in 48 healthy sheep, filmed at one preoperative and three postoperative moments, before and after rescue analgesia and 24 hours after. The videos were randomised and assessed twice by four evaluators, with a one-month interval between evaluations. Statistical analysis was performed using R software and differences were considered significant when p <0.05. Based on the multiple association, a unidimensional scale was adopted. The intra- and inter-observer reliability ranged from moderate to very good (intraclass correlation coefficient ≥ 0.53). The scale presented Spearman correlations > 0.80 with the numerical, simple descriptive, and visual analogue scales, and a correlation of 0.48 with the facial expression scale. According to the mixed linear model, the scale was responsive, due to the increase and decrease in pain scores of all items after surgery and analgesic intervention, respectively. All items on the scale demonstrated an acceptable Spearman item-total correlation (0.56-0.76), except for appetite (0.25). The internal consistency was excellent (Cronbach's α = 0.81) and all items presented specificity > 0.72 and sensitivity between 0.61-0.90, except for appetite. According to the Youden index, the cut-off point was ≥ 4 out of 12, with a diagnostic uncertainty zone of 4 to 5. The area under the curve > 0.95 demonstrated the excellent discriminatory capacity of the instrument. In conclusion, the Unesp-Botucatu pain scale in sheep submitted to laparoscopy is valid, reliable, specific, sensitive, with excellent internal consistency, accuracy, discriminatory capacity, and a defined cut-off point.

Comparison of two surgical approaches for Laparoscopic Ovum Pick Up in Ewes / Comparação de duas abordagens cirúrgicas para aspiração folicular laparoscópica em ovelhas

The present study had as an aim to evaluate a right lateral access as an alternative method to laparoscopic ovum pick-up (LOPU) in sheep. Twenty-four Santa Ines ewes were randomly assigned in two groups with twelve animals each: RLD - positioned in right lateral decubitus, with 10º head-down tilt; and DD - positioned in dorsal decubitus with 35º head-down tilt. The following parameters were evaluated every 10 minutes during the procedure: total surgical time (ST), visualized follicles (VF), aspirated follicles (AF), recovered oocytes (RO), mean arterial pressure (MAP), heart rate (HR), respiratory rate (fR) and end tidal CO2 pressure (EtCO2). Pre and postoperative arterial hemogasometry parameters (PaO2, PaCO2, pH, CHCO3 and BE) were also evaluated; and serum fibrinogen levels (SFL) on postoperative period. The values of VF, AF, RO, fR, PaO2, pH, CHCO3, BE and SFL were similar between groups, although ST, HR, MAP, EtCO2 and PaCO2 were higher in LG. Regarding operative periods, PaO2 and pH were lower after surgery (PaO2: 79.1±16.4; 79.2±11.7mmHg; pH: 7.30±0.09; 7.32±0.08) in both groups when compared to preoperative (PaO2: 80.1±14.3; 83.4±10.5 mmHg; pH: 7.38±0.05; 7.39±0.05) while PaCO2 (43.6±4.6; 41.9±5.4mmHg) and CHCO3 (22.8±1.5; 22.7±3.0mmol/L) increased (PaCO2: 54.3±10.9; 46.9±6.3mmHg; CHCO3: 24.8±3.4; 24.4±2.7mmol/L) postoperative. This alternative decubitus presented is a viable procedure and did not differ in oocyte recovery rates in ewes. However, entails cardiorespiratory major alterations compared to conventional procedure, making its practical applicability limited.(AU)

O presente estudo teve como objetivo avaliar o acesso lateral direito como um método alternativo para a recuperação de oócitos por laparoscopia (LOPU) em ovelha. Vinte e quatro ovelhas Santa Inês foram distribuídas aleatoriamente em dois grupos com 12 animais: grupo RLD - posicionado em decúbito lateral direito, cefalodeclive com 10º de inclinação; grupo DD - posicionado em decúbito dorsal em cefalodeclive, inclinação de 35º. Foram avaliados, a cada 10 minutos, durante o procedimento cirúrgico: tempo total da cirurgia (ST), folículos visualizados (VF), folículos aspirados (AF), oócitos recuperados (RO), pressão arterial média (MAP), frequência cardíaca (FC), frequência respiratória (fR) e pressão final de CO2 (EtCO2). Também foram avaliados os parâmetros de hemogasometria arterial pré-operatória e pós-operatória (PaO2, PaCO2, pH, CHCO3 e BE), bem como os níveis séricos de fibrinogênio (SFL) no período pós-operatório. Os valores de VF, AF, RO, fR, PaO2, pH, CHCO3, BE e SFL foram semelhantes entre os grupos, embora ST, FC, MAP, EtCO2 e PaCO2 tenham sido maiores em RLD. Os parâmetros PaO2 e pH foram menores após a cirurgia (PaO2: 79,1±16,4; 79,2±11,7mmHg; pH: 7,30±0,09; 7,32±0,08) em ambos os grupos em relação ao momento pré-cirúrgico (PaO2: 80,1±14,3; 83,4±10,5mmHg; pH: 7,38±0,05; 7,39±0,05), enquanto PaCO2 (43,6±4.6; 41,9±5,4mmHg) e CHCO3 (22,8±1,5; 22,7±3.0mmol/L) aumentaram (PaCO2: 54,3±10,9; 46,9±6,3mmHg; CHCO3: 24,8±3,4; 24,4±2,7mmol/L) após a cirurgia. O decúbito lateral é uma alternativa viável para LOPU e não apresenta diferença para a taxa de recuperação oocitária em ovelhas. No entanto, promove alterações cardiorrespiratórias em comparação com o decúbito dorsal, tornando a sua aplicabilidade prática limitada.(AU)

Effect of three opioid-based analgesic protocols on the perioperative autonomic-mediated cardiovascular response in sheep.

Few reports evaluate the clinical effects of opioids in sheep during experimental surgical procedures. Catecholamine-mediated haemodynamic changes resulting from surgical noxious stimulation are blunted by opioids. The aim of this study was to evaluate the efficacy of three opioid-based analgesic protocols in avoiding a 20% increase in heart rate (HR) and/or mean arterial blood pressure (MAP) during experimental intervertebral disk nucleotomy in sheep. Eighteen female Brogna sheep were anaesthetized with propofol and maintained with a fixed end-tidal isoflurane concentration of 1.5 ± 0.1%. Sheep were assigned to one of three groups that intravenously received methadone 0.3 mg/kg (group M), fentanyl 2 µg/kg followed by 10 µg/kg/h (group F), or buprenorphine 10 µg/kg and 30 minutes later ketamine 1 mg/kg followed by 5 mg/kg/h (group BK). Intravenous fentanyl at 2 µg/kg would have been used for rescue analgesia in case HR and/or MAP had increased. During surgery, HR and MAP values did not increase over 20% in all groups. All animals maintained the percentage change between -4 and 7% for both variables; only one sheep in group BK had an increase in MAP superior to 20% after ketamine administration before surgical stimulation. In group M, HR decreased over time and in group BK, MAP tended to increase during surgery. All the opioid-based protocols tested were able to control the cardiovascular response to noxious stimulation in sheep undergoing spinal surgery, although ketamine may have represented a confounding factor.

Volatile Profile of Grilled Lamb as Affected by Castration and Age at Slaughter in Two Breeds.

The aim of this study was to assess the effect of castration and slaughter age on the volatile profile of cooked meat from Scottish Blackface (SB) and Texel × Scottish Blackface (T × SB) lambs. M. longissimus thoracis et lumborum was sampled at slaughter and subjected to volatile analysis by SPME-GC-MS. Rams had higher relative proportions, expressed as relative abundance (RA), in lipid oxidation products while castrates had higher RA in pyrazines and benzenoid compounds. There was no consistent age effect on the RA of volatiles, although rams in November and January had a different volatile profile to castrates. There were higher proportions of free branched-chain fatty acids in muscle from SB compared to T × SB lambs. Overall, the results showed that production factors affected the volatile profile of cooked lamb meat which may explain differences in lamb flavor. PRACTICAL APPLICATION: Lamb meat has a characteristic flavor which, according to the evidence to date, may be influenced by farm production factors like gender or slaughter age. Our results showed variations in the proportions of some flavor compounds in cooked lamb between rams and castrated lambs while an increase in slaughter age did not have a consistent effect on proportions of compounds.

Differential effects of propofol and isoflurane on the relationship between EEG Narcotrend index and clinical stages of anaesthetic depth in sheep undergoing experimental cardiac surgery.

The electroencephalogram (EEG) Narcotrend index (NI) has been shown to improve anaesthetic depth monitoring in isoflurane-anaesthetised sheep. Data obtained from 13 anaesthetised juvenile female sheep were analysed retrospectively in order to assess the relationship between clinical stages of anaesthesia (CS) and NI during both propofol and isoflurane anaesthesia. Polynomial regression analysis revealed no significant association between CS and NI for propofol (R = 0.374, R(2) = 0.140, P = 0.403) but for isoflurane anaesthesia (R = 0.548, R(2) = 0.301, P = 0.010) there was a significant relationship. Furthermore, a strong correlation existed between end-tidal isoflurane concentration (ISOET) and CS (r = -0.463, P = 0.008). A combination of assessment of clinical signs and analogous EEG patterns is recommended during propofol anaesthesia.

The sheep as a model for healing studies after partial nephrectomy.

BACKGROUND: The pig has been considered the best model for renal surgery. However, recent research has demonstrated that the kidney of pigs heals differently from that of humans. The objective of this study was to evaluate sheep as an alternative animal model for studying collecting system healing after laparoscopic partial nephrectomy. MATERIALS AND METHODS: The caudal pole of the left kidney was removed from eight female adult domestic sheep using laparoscopic partial nephrectomy. Monopolar energy was used for hemostasis only in the parenchyma, avoiding coagulation near the collecting system, which was left opened. After 14 d, all animals were euthanized, and their left kidney was removed. Serum levels of urea and creatinine were assessed preoperatively and postoperatively (on days 2, 6, 10, and 14), and peritoneal fluid samples were collected during necropsy for urea and creatinine evaluation. An ex vivo retrograde pyelogram was performed, and a retrograde injection of methylene blue ink was administered to evaluate urinary leakage. Samples from the operated pole were analyzed using histologic methods. RESULTS: During necropsy, an urinoma surrounding the operated kidney was observed in one animal. Peritoneal fluid levels of urea and creatinine were elevated. Retrograde pyelograms exhibited contrast-medium extravasation through the operated pole in all kidneys. The opened collecting system was also confirmed by methylene blue ink injection. The operated pole was covered by collagenous tissue and adhered to adjacent organs. CONCLUSIONS: Sheep should be considered as an adequate experimental model for research on collecting system healing after partial nephrectomy.

Post-operative echocardiographic evaluation of bioprosthetic mitral valve implantation in sheep.

The ovine model is generally considered to be the best for testing bioprosthetic heart valve durability. Although echocardiography is the method of choice for the interim evaluation of the valve, literature on sheep echocardiography is scarce. Within the context of a study on treatment of pericardial heart valve prostheses, 19 adolescent sheep underwent transthoracic echocardiography six days after mitral implantation of bioprosthetic valves. Echocardiographic examination was performed under mild anesthesia and animals were put in a right lateral decubitus position. Four images were obtained: right parasternal long axis four and five chamber views, right parasternal long axis view with left ventricular outflow, and right parasternal short axis view through the mitral valve. We measured aortic annulus and velocity time integral over the aortic valve to determine stroke volume, cardiac output and cardiac index. The mitral valve was evaluated through color Doppler imaging for valvular and paravalvular leakages. Pulsed wave spectral Doppler was used for the measurement of velocities, pressures and velocity time integrals. For the evaluation of valve stenosis deceleration time and pressure half-time were determined. Effective orifice area of the mitral valve was derived. And, although not measured, other structures could clearly be visualized: right and left ventricle and atrium, wall thicknesses, tricuspid valve. This study shows that echocardiography in sheep is feasible, and that right parasternal images, obtained in animals in a right lateral decubitus position, are well qualified for the interim evaluation of bioprosthetic valves implanted in the mitral position. Besides the implanted valve, other cardiac structures like atria and ventricles can be visualized and evaluated.

Modification of a method for cannulation of the cisterna magna in sheep to enable chronic collection of cerebrospinal fluid.

A method is described for chronic cannulation of the cisterna magna to enable repeated sampling of cerebrospinal fluid from conscious, ambulatory sheep by means of a flexible vinyl tube. Ease of sampling and duration of cannula patency are similar to those obtained with rigid, metal cannulae, but this modified method minimizes the degree of surgical intervention, and possible trauma, occurring during placement of the cannula.

Improvement in renal hemodynamics following combined angiotensin II infusion and AT1R blockade in aged female sheep following fetal unilateral nephrectomy.

Renin-angiotensin system (RAS) is a powerful modulator of renal hemodynamic and fluid homeostasis. Up-regulation in components of intra-renal RAS occurs with ageing. Recently we reported that 2 year old uninephrectomised (uni-x) female sheep have low renin hypertension and reduced renal function. By 5 years of age, these uni-x sheep had augmented decrease in renal blood flow (RBF) compared to sham. We hypothesised that this decrease in RBF in 5 year old uni-x sheep was due to an up-regulation in components of the intra-renal RAS. In this study, renal responses to angiotensin II (AngII) infusion and AngII type 1 receptor (AT1R) blockade were examined in the same 5 year old sheep. We also administered AngII in the presence of losartan to increase AngII bioavailability to the AT2R in order to understand AT2R contribution to renal function in this model. Uni-x animals had significantly lower renal cortical content of renin, AngII (∼40%) and Ang 1-7 (∼60%) and reduced cortical expression of AT1R gene than sham animals. In response to both AngII infusion and AT1R blockade via losartan, renal hemodynamic responses and tubular sodium excretion were significantly attenuated in uni-x animals compared to sham. However, AngII infusion in the presence of losartan caused ∼33% increase in RBF in uni-x sheep compared to ∼14% in sham (P<0.05). This was associated with a significant decrease in renal vascular resistance in the uni-x animals (22% vs 15%, P<0.05) without any changes in systemic blood pressure. The present study shows that majority of the intra-renal RAS components are suppressed in this model of low renin hypertension. However, increasing the availability of AngII to AT2R by AT1R blockade improved renal blood flow in uni-x sheep. This suggests that manipulation of the AT2R maybe a potential therapeutic target for treatment of renal dysfunction associated with a congenital nephron deficit.

Experimentally induced cam impingement in the sheep hip.

Sheep hips have a natural non-spherical femoral head similar to a cam-type deformity in human beings. By performing an intertrochanteric varus osteotomy, cam-type femoro-acetabular impingement (FAI) during flexion can be created. We tested the hypotheses that macroscopic lesions of the articular cartilage and an increased Mankin score (MS) can be reproduced by an experimentally induced cam-type FAI in this ovine in vivo model. Furthermore, we hypothesized that the MS increases with longer ambulatory periods. Sixteen sheep underwent unilateral intertrochanteric varus osteotomy of the hip with the non-operated hip as a control. Four sheep were sacrificed after 14, 22, 30, and 38-weeks postoperatively. We evaluated macroscopic chondrolabral alterations, and recorded the MS, based on histochemical staining, for each ambulatory period. A significantly higher prevalence of macroscopic chondrolabral lesions was found in the impingement zone of the operated hips. The MS was significantly higher in the acetabular/femoral cartilage of the operated hips. Furthermore, these scores increased as the length of the ambulatory period increased. Cam-type FAI can be induced in an ovine in vivo model. Localized chondrolabral degeneration of the hip, similar to that seen in humans (Tannast et al., Clin Orthop Relat Res 2008; 466: 273-280; Beck et al., J Bone Joint Surg Br 2005; 87: 1012-1018), can be reproduced. This experimental sheep model can be used to study cam-type FAI.

Biocompatibility assessment of a long-term wearable artificial pump-lung in sheep.

The purpose of this study was to assess the biocompatibility of a newly developed long-term wearable artificial pump-lung (APL) in a clinically relevant ovine animal model. The wearable APL device was implanted in five sheep through left thoracotomy. The device was connected between the right atrium and pulmonary artery and evaluated for 30 days. Three sheep were used as the sham control. Platelet activation was assessed by measuring platelet surface P-selectin (CD62P) expression with flow cytometry and plasma soluble P-selectin with an enzyme-linked immunosorbent assay. Thrombotic deposition on the device components and hollow fiber membranes were analyzed with digital imaging and scanning electron microscopy. Surface P-selectin of the APL and sham groups changed significantly over the study period, but without significant differences between the two groups. Soluble P-selectin for the two groups peaked in the first 24 h after the surgery. Soluble P-selectin of the APL group remained slightly elevated over the study period compared to the presurgical baseline value and was slightly higher compared to that of the sham group. Plasma free hemoglobin remained in the normal ranges in all the animals. In spite of the surgery-related alteration in laboratory tests and elevation of platelet activation status, the APL devices in all the animals functioned normally (oxygen transfer and blood pumping) during the 30-day study period. The device flow path and membrane surface were free of gross thrombus. Electron microscopy images showed only scattered thrombi on the fibers (membrane surface and weft). In summary, the APL exhibited excellent biocompatibility. Two forms of platelet activation, surgery-related and device-induced, in the animals implanted with the wearable APL were observed. The limited device-induced platelet activation did not cause gross thrombosis and impair the long-term device performance.

The preclinical sheep model of high tibial osteotomy relating basic science to the clinics: standards, techniques and pitfalls.

PURPOSE: To develop a preclinical large animal model of high tibial osteotomy to study the effect of axial alignment on the lower extremity on specific issues of the knee joint, such as in articular cartilage repair, development of osteoarthritis and meniscal lesions. Preoperative planning, surgical procedure and postoperative care known from humans were adapted to develop a HTO model in the adult sheep. METHODS: Thirty-five healthy, skeletally mature, female Merino sheep between 2 and 4 years of age underwent a HTO of their right tibia in a medial open-wedge technique inducing a normal (group 1) and an excessive valgus alignment (group 2) and a closed-wedge technique (group 3) inducing a varus alignment with the aim of elucidating the effect of limb alignment on cartilage repair in vivo. Animals were followed up for 6 months. RESULTS: Solid bone healing and maintenance of correction are most likely if the following surgical principles are respected: (1) medial and longitudinal approach to the proximal tibia; (2) biplanar osteotomy to increase initial rotatory stability regardless of the direction of correction; (3) small, narrow but long implant with locking screws; (4) posterior plate placement to avoid slope changes; (5) use of bicortical screws to account for the brittle bone of the tibial head and to avoid tibial head displacement. CONCLUSION: Although successful high tibial osteotomy in sheep is complex, the sheep may--because of its similarities with humans--serve as an elegant model to induce axial malalignment in a clinically relevant environment, and osteotomy healing under challenging mechanical conditions.

Effect of administration of platelet-rich plasma in early phases of distraction osteogenesis: an experimental study in an ovine femur model.

BACKGROUND: It has been suggested that platelet-rich plasma (PRP) might enhance bone formation. The aim of this study was to quantify the effect of PRP administered in the early phases of distraction osteogenesis in an ovine femur model. METHODS: Twenty sheep aged 4 months underwent osteotomy of the femoral diaphysis followed by distraction osteogenesis. The sheep were divided into two groups of 10. One group received three injections of PRP on days 0, 10, and 20 of the procedure (PRP group) and the other received no additional treatment (control group). The results were evaluated by computed tomography (CT) and histology on completion of distraction osteogenesis (day 40). CT evaluation included measurement of the callus and bone density. Femur size was also measured proximally and distally. Histological evaluation was used to quantify osteoblasts, osteoclasts, vascular lumens, and trabecular maturity by zones and to calculate trabecular bone, fibrous tissue, and cartilage ratios. RESULTS: Radiological and histological evaluation of the regenerate bone showed no significant differences between the PRP group and the control group for any of the variables analysed. The only significant difference detected was a wider femur (increased diaphyseal thickness) at the proximal and distal levels in the PRP group. CONCLUSION: We found no radiological or histological evidence that the administration of PRP in the early phases of distraction osteogenesis enhances bone formation.

Tissue engineering for total meniscal substitution: animal study in sheep model--results at 12 months.

The aim of the study was to investigate the use of a hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Eighteen skeletally mature sheep were treated. The animals were divided into three groups: cell-free scaffold, scaffold seeded with autologous chondrocytes, and meniscectomy alone. The implant was sutured to the capsule and to the meniscal ligament. At a 12-month gross assessment, histology and histomorphometry were used to assess the meniscus implant, knee joint, and osteoarthritis development. All implants showed excellent capsular ingrowth at the periphery. The implant gross assessment showed significant differences between cell-seeded and cell-free groups (p=0.011). The histological analysis indicated a cellular colonization throughout the implanted constructs. Avascular cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Joint gross assessment showed that sheep treated with scaffold implantation achieved a significant higher score than those underwent meniscectomy (p<0.0005), and the Osteoarthritis Research Society International score showed that osteoarthritic changes were significantly less in the cell-seeded group than in the meniscectomy group (p=0.047), even though results were not significantly superior to those of the cell-free scaffold. Seeding of the scaffold with autologous chondrocytes increases its tissue regeneration capacity, providing a better fibrocartilaginous tissue formation. The study suggests the potential of the novel hyaluronic acid/polycaprolactone scaffold for total meniscal substitution, although this approach has to be further improved before being applied into clinical practice.

A novel ovine ex vivo arteriovenous shunt model to test vascular implantability.

The major objective of successful development of tissue-engineered vascular grafts is long-term in vivo patency. Optimization of matrix, cell source, surface modifications, and physical preconditioning are all elements of attaining a compatible, durable, and functional vascular construct. In vitro model systems are inadequate to test elements of thrombogenicity and vascular dynamic functional properties while in vivo implantation is complicated, labor-intensive, and cost-ineffective. We proposed an ex vivo ovine arteriovenous shunt model in which we can test the patency and physical properties of vascular grafts under physiologic conditions. The pressure, flow rate, and vascular diameter were monitored in real-time in order to evaluate the pulse wave velocity, augmentation index, and dynamic elastic modulus, all indicators of graft stiffness. Carotid arteries, jugular veins, and small intestinal submucosa-based grafts were tested. SIS grafts demonstrated physical properties between those of carotid arteries and jugular veins. Anticoagulation properties of grafts were assessed via scanning electron microscopy imaging, en face immunostaining, and histology. Luminal seeding with endothelial cells greatly decreased the attachment of thrombotic components. This model is also suture free, allowing for multiple samples to be stably processed within one animal. This tunable (pressure, flow, shear) ex vivo shunt model can be used to optimize the implantability and long-term patency of tissue-engineered vascular constructs.

Pain in lambs castrated at 2 days using novel smaller and tighter rubber rings without and with local anaesthetic.

Without effective pain relief, rubber ring castration of lambs is acutely painful and can also produce chronic pain. The potential of novel, smaller rubber rings to reduce this pain substantially has been investigated. Three groups of eight 2-3 day old lambs, were castrated either with conventional rubber rings (cRR), or novel smaller rubber rings without (nRR) and with local anaesthetic treatment (nRR+La). Behavioural responses and chronic lesions indicative of pain were compared. No major reductions in behavioural responses were produced by castration with nRRs compared with cRRs, but injection of local anaesthetic did reduce these responses significantly. Lambs in all groups developed chronic inflammatory lesions but behavioural evidence of chronic pain was rarely seen. After 28 days, 6/8 necrotic scrotums had been shed by nRR+La; 4/8 by nRR and 2/8 by cRR lambs. The dimensions of rubber rings on necrotic scrotums, suggest that cRRs contracted to their un-stretched size before being shed, whereas nRRs did not. It is concluded that neither of these rubber rings exerted sufficient pressure on trapped nerves to produce anaesthesia rapidly and this may be explained by the physical properties of the tissues at the neck of the scrotum which support the ring.