Effects of Hypoxia and Chitosan on Equine Umbilical Cord-Derived Mesenchymal Stem Cells.

Chitosan opens new perspectives in regenerative medicine as it enhances the properties of mesenchymal stem cells (MSCs) through formation of spheroids. Hypoxia has also been proposed to enhance stemness and survival of MSCs after in vivo implantation. These characteristics are relevant to the development of an off-the-shelf source of allogenic cells for regenerative therapy of tendinopathies. Umbilical cord-derived MSCs (UCM-MSCs) offer an abundant source of immature and immunoprivileged stem cells. In this study, equine UCM-MSCs (eqUCM-MSCs) conditioned for 3 and 7 days on chitosan films at 5% oxygen were compared to eqUCM-MSCs under standard conditions. Equine UCM-MSCs formed spheroids on chitosan but yielded 72% less DNA than standard eqUCM-MSCs. Expression of Sox2, Oct4, and Nanog was 4 to 10 times greater in conditioned cells at day 7. Fluorescence-labeled cells cultured for 7 days under standard conditions or on chitosan films under hypoxia were compared in a bilateral patellar tendon defect model in rats. Fluorescence was present in all treated tendons, but the modulus of elasticity under tension was greater in tendons treated with conditioned cells. Chitosan and hypoxia affected cell yield but improved the stemness of eqUCM-MSCs and their contribution to the healing of tissues. Given the abundance of allogenic cells, these properties are highly relevant to clinical applications and outweigh the negative impact on cell proliferation.

Biomechanical comparison of strategies to adjust axial stiffness of a hybrid fixator.

OBJECTIVES: To evaluate strategies for increasing the axial stiffness of a hybrid external bone fixator. MATERIALS AND METHODS: Type Ia hybrid fixators, consisting of a uniplanar linear component connected to a circular ring, were tested in displacement controlled loading in axial compression. The basic hybrid construct was modified to explore strategies considered to increase fixator stiffness including: decreasing ring diameter, increasing ring thickness, adding pins to the ring fixation, and adding struts between the ring and vertical post components of the device. Stiffness in the initial phase of loading was compared between the groups. RESULTS: The addition of a single diagonal bar between the ring and linear connecting rail did not significantly improve the stiffness of constructs. However, the addition of two half-pins to the ring, the addition of two struts between the ring and linear connecting rail, or decreasing the internal ring diameter from 115 to 85 mm progressively increased the stiffness of the frame. The most effective strategy consisted of increasing the thickness of the ring from 6 to 12 mm, thereby increasing the stiffness of the control frame by 335%. CLINICAL SIGNIFICANCE: Modulating the ring thickness, adding two struts between the ring and linear connecting rail, and reducing the ring diameter appear to be the most effective, simple, and clinically versatile ways to increase axial stiffness, most likely due to their impact on reducing ring bending.

The rationale behind novel bone grafting techniques in small animals.

Autograft is considered ideal for grafting procedures, providing osteoinductive growth factors, osteogenic cells, and an osteoconductive scaffold. Limitations, however, exist regarding donor site morbidity and graft availability. Although allograft provides an osteoconductive matrix with some osteoinductivity, its availability is limited. To achieve optimal bone graft properties, researchers are developing new materials with the goal of designing synthetics as close to autograft as possible while still facilitating their clinical use. However, the constant evolution of internal fixation stimulates the search for growth factors and cells which could stimulate bone healing.

Double-blind placebo-controlled trial of adjuvant pamidronate with palliative radiotherapy and intravenous doxorubicin for canine appendicular osteosarcoma bone pain.

BACKGROUND: Canine osteosarcoma (OSA) causes focal malignant osteolysis leading to severe pain. Despite the documented efficacy of radiotherapy or IV aminobisphosphonates for managing cancer bone pain, their potential combined therapeutic value has not been reported in OSA-bearing dogs. HYPOTHESIS: Pamidronate combined with standardized palliative therapy will improve pain control and bone biologic effects in OSA-bearing dogs. ANIMALS: Fifty dogs with appendicular OSA treated with standardized palliative therapy and either pamidronate or sterile saline. METHODS: Randomized, prospective, double-blinded, placebo-controlled study. Treatment responses for dogs receiving standardized palliative therapy with (n = 26) or without (n = 24) adjuvant pamidronate were serially evaluated for changes in subjective pain scores, urine N-telopeptide (NTx) excretion, primary tumor relative bone mineral density (rBMD), and computerized pressure platform gait analysis. RESULTS: Median duration of subjective pain relief for dogs treated with adjuvant pamidronate or placebo was 76 and 75 days, respectively (P= .39). Forty percent (20/50; pamidronate [11/26] and placebo [9/24]) of dogs experienced durable analgesia, defined by pain alleviation > or =112 days. For patients achieving durable pain control, dogs treated with pamidronate achieved greater reductions in NTx excretion and larger increases in rBMD compared with placebo controls. Changes in peak vertical force assessed by computerized pressure platform gait analysis correlated with pain alleviation in OSA-bearing dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Combining pamidronate with standardized palliative therapy is safe, but does not clearly improve pain alleviation. However, in dogs achieving durable pain control, adjuvant pamidronate appears to decrease focal bone resorption in the local tumor microenvironment.

A comparison of five different bone resorption markers in osteosarcoma-bearing dogs, normal dogs, and dogs with orthopedic diseases.

BACKGROUND: Various bone resorption markers in humans are useful for supporting the diagnosis of malignant skeletal pathology, with certain bone resorption markers appearing to be more discriminatory for detecting cancer-induced osteolysis than others. Canine osteosarcoma (OSA) is characterized by focal bone destruction, but a systematic investigation for determining which bone resorption marker best supports the diagnosis of OSA in dogs has not been reported. HYPOTHESIS: Dogs with OSA will have increased concentrations of bone resorption markers compared with healthy dogs and dogs with orthopedic disorders. Differences will exist among various bone resorption markers for their ability to support the diagnosis of malignant osteolysis in dogs with OSA. ANIMALS: Single time point, cross-sectional, cohort study including dogs with OSA (n = 20) or orthopedic disorders (n = 20) and healthy dogs (n = 22). METHODS: Basal concentrations of urine and serum N-telopeptide (NTx), urine and serum C-telopeptide (CTx), and urine deoxypyridinoline (DPD) were compared among all 3 groups. RESULTS: Compared with healthy dogs and dogs with orthopedic disorders, urine NTx, serum NTx, and serum CTx concentrations were significantly increased in dogs with OSA. For urine NTx and serum NTx, the calculated lower and upper 95% confidence limits in dogs with OSA did not overlap with dogs diagnosed with orthopedic disorders or healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Of the markers evaluated in this study, urine NTx and serum NTx appear to be the most discriminatory resorption markers supporting the diagnosis of focal malignant osteolysis in dogs with OSA.

In vivo behavior of zirconia hydroxyapatite (ZH) ceramic implants in dogs: a clinical, radiographic, and histological study.

The main goal of this study is to evaluate potential applications of two zirconia-hydroxyapatite composites, Z4H6 and Z6H4, as bone substitutes. Composite plugs were implanted into the distal femoral metaphysis and also onto the longissimus dorsi of 18 adult mixed-breed dogs in order to assess in vivo biocompatibility by immediate clinical and radiographic evaluation 30, 90, and 120 days after implantation. Radiographic examination revealed radiolucency on the defect site. However, a progressive increase in bone density was observed over time, reaching a radiopacity similar to that of bone 120 days after implantation. Histological study revealed that a thin layer of fibroblasts was observed at the implant-bone interface in addition to osteoblastic activity 30 days after implantation, whereas bone neoformation around the implants was detected for the subsequent implantation times (90 and 120 days). Otherwise, the histological evaluation of the implant-muscle interface showed the presence of an initially thick fibrous tissue layer 30 days after implantation, which decreased with longer investigation times (90 and 120 days). The numbers of plasmocytes, lymphocytes, and macrophages gradually reduced as a function implantation time, being completely absent 120 days after implantation with a resulting complete osteointegration process. The zirconia phase content did not affect the bioactive behavior of the implants investigated and did not induce bone formation when implanted into muscle either.

Impaction grafting with morsellised allograft and tricalcium phosphate-hydroxyapatite: incorporation within ovine metaphyseal bone defects.

An ovine model was used to investigate the in vivo properties of impacted tricalcium phosphate-hydroxyapatite (TCP-HA) aggregates, varying in chemical composition (ratio of TCP to HA) and particle size distribution (8 versus 3 particle size ranges). All pellets were impacted to a standard compactive effort. Eight sheep underwent implantation of pellets in 4 metaphyseal defects in both rear limbs. Treatment groups consisted of: (1) allograft (clinical control). (2) 50/50 allograft/80% HA/20% TCP in 8 particle size ranges, (3) 50/50 allograft/80% TCP/20% HA in 8 sizes and (4) 50/50 allograft/80% HA/20% TCP in only 3 sizes of particles. Healing of defects was evaluated at 14 weeks with computed tomography, histology and histomorphometry. The computer tomography (CT) density measured in all defects containing synthetic agents was higher than in defects filled with allograft alone (p<0.01). Defects containing 8 sizes of 80% HA/ 20% TCP granules (group 2) achieved lower histological scores and contained less bone than the clinical control (p<0.05), whereas groups 3 and 4 did not differ from the control. Although all synthetic agents were osteoconductive, our results suggest that increasing the ratio of TCP over HA and limiting the number of particle size ranges to 3 instead of 8 improve the performance of impacted aggregates as graft expanders. Evaluation under loading conditions of morsellised allograft expanded with 80% TCP/20% HA (BoneSave) in 3 particle size ranges is warranted.

Early dissolution of a morsellised impacted silicate-free bioactive glass in metaphyseal defects.

The purpose of this study was to evaluate the biological properties of idealized, morsellised impacted Corglaes, a soluble silicate-free glass. Twenty-two sheep underwent implantation of pellets in six metaphyseal defects in both rear limbs. Particle size distribution of all aggregates (except the autograft) approached the ideal logarithmic grading line and all implants were impacted to a standard compactive effort. Treatment groups consisted of (1) empty defect (negative control), (2) autograft (positive control), (3) allograft (clinical control), (4) allograft idealized with Corglaes, (5) 50/50 allograft/Corglaes and (6) Corglaes. Defects were sealed with polymethylmethacrylate. Healing of the defects was evaluated at 7 weeks (n=6) and 14 weeks (n=16) with computed tomography, histology, and histomorphometry. Remnants of Corglaes were found within one of the defects 7 weeks after implantation. Defects filled with mixtures containing 50% or 100% Corglaes were less dense, contained less bone and more fibrous tissue than defects with allograft, autograft, or allograft idealized with Corglaes. Allograft idealized with Corglaes may be considered for impaction grafting in revision hip arthroplasty, as well as local delivery of antibiotics. However, further studies and potential revision of the agent are required before mixtures containing concentrations of Corglaes equal to or higher than 50% can be recommended.

An ovine model to evaluate the biologic properties of impacted morselized bone graft substitutes.

The objectives of this study were to develop and evaluate a bone-defect model to study the biologic behavior of biomaterials being considered for impaction grafting in revision hip arthroplasty. A miniature impactor was designed to produce pellets of aggregates at a standard compactive effort. In Phase 1, 22 sheep underwent implantation of pellets into six metaphyseal defects in both rear limbs. In Phase 2, eight sheep underwent surgical implantation of four pellets in metaphyseal defects. Defects were sealed with polymethylmethacrylate in both phases. Healing of the defects was evaluated at 7 weeks (Phase 1, n = 11) and 14 weeks (n =19) with computed tomography, histology, and histomorphometry. Complications in Phase 1 included four femoral fractures and migration of cement seals (18/102 defects). No complications occurred in Phase 2. Whereas no difference was found between left and right limbs, osteogenesis and incorporation of biomaterials varied among implantation sites. For comparison of grafting materials, treatment site allocations were randomized according to a Latin square design. This model allows evaluation of several impacted aggregates (including large particles) in the same animal. It is particularly suitable for analyzing the biologic properties of grafting materials prior to evaluation under loading conditions.

Evaluation of a hemostasis model for teaching ovariohysterectomy in veterinary surgery.

OBJECTIVE: To evaluate the efficacy of a reusable plastic model mimicking the anatomy and hemodynamics of the canine female genital tract for teaching basic surgical skills and ovariohysterectomy. SAMPLE POPULATION: 40 veterinary students of the class of 1998. STUDY DESIGN: Prospective study. METHODS: Students'confidence level and experience in private practice was evaluated via questionnaire before training. Students in 2 groups performed an ovariohysterectomy on cadavers (group C, n = 20) or on the model (Group M, n = 20) for 2 hours. Students' psychomotor and basic surgical skills were objectively assessed by the following tests: ligation of a foam cylinder, passing a needle through the eyelets of an electronic suture board, and ligating latex tubing. Results were compared before and after training and within and between groups. The ability of students to perform an ovariohysterectomy in a live dog after training was compared between groups with a scoring system. RESULTS: Students in both groups had similar surgical experience and basic skills before training. The results of the psychomotor and basic surgical skills tests were better in group M after training than group C. The improvement of each student in performing these tasks also increased when students were trained with the model. Scores assigned to students performing an ovariohysterectomy in a live dog were higher in group M (31.45 +/- 1.15) than in group C (20.7 +/- 1.42). CONCLUSION: The model was more effective than cadavers in teaching basic surgical skills and ovariohysterectomy in dogs.

Use of a mucosal advancement flap for the treatment of nasopharyngeal stenosis in a cat.

A three-and-a-half-year-old male neutered domestic short-haired cat was presented with a three-year history of stertor and intermittent open-mouth breathing. No airflow was detected from either nostril when checked using a cold slide test. Oropharyngeal swabs were positive for calicivirus, while skull radiographs were suggestive of a dorsal deviation of the soft palate. The diagnosis of nasopharyngeal stenosis was confirmed via cannulation of the nasal passages and direct examination of the oropharynx under general anaesthesia. A midline approach through the soft palate was used to excise the adhesions. The resulting defect was reconstructed by advancement of a mucosal flap elevated from the dorsal nasopharynx and laryngopharynx. The cat was free of clinical signs 28 months later.

Resolution of paraneoplastic alopecia following surgical removal of a pancreatic carcinoma in a cat.

A 13-year-old female neutered domestic longhaired cat was presented with a five-month history of progressive weight loss and bilaterally symmetrical alopecia of the ventrum, limbs and perineum. The alopecic skin had a shiny appearance and hair in the non-alopecic areas was easily epilated. Fine needle aspirate cytology of a palpable cranial abdominal mass revealed it to be of epithelial or glandular origin. A pancreatic mass was excised by left pancreatectomy during exploratory laparotomy, and histopathology and skin biopsies confirmed a diagnosis of pancreatic carcinoma with concurrent paraneoplastic alopecia. No evidence of metastases was found on liver and lymph node biopsies. At re-examination 10 weeks after surgery, the hair had fully regrown. Skin signs recurred after 18 weeks and metastatic spread of the tumour was confirmed on postmortem examination. This case confirms that paraneoplastic alopecia associated with internal malignancies is a potentially reversible process if the internal neoplasm is excised.

Biomechanical properties of canine corticocancellous bone frozen in normal saline solution.

Eleven pairs of canine metacarpal bones, 10 pairs of metatarsal bones, and 7 pairs of ribs were harvested cleanly and prepared for banking at -20 C for 1 year. One bone of each pair was randomly assigned to 1 type of storage: plastic pack vs immersion in a normal solution of sodium chloride. The contralateral bone was assigned to the opposite treatment. Six pairs of metacarpal bones and 5 pairs of metatarsal bones were tested in torsion to failure. No significant difference was found within pairs. All ribs, 5 pairs of metacarpal bones, and 5 pairs of metatarsal bones were loaded in 4-point bending to failure. The energy absorbed at failure and the ultimate displacement of ribs and metacarpal and metatarsal bones were increased by 25 to 30% and 18 to 24%, respectively, when the bones were frozen in isotonic saline solution. Corticocancellous grafts frozen in normal saline solution are biomechanically less fragile and brittle than grafts stored in plastic without saline solution.