Calcium phosphate ceramics combined with rhBMP6 within autologous blood coagulum promote posterolateral lumbar fusion in sheep.

Posterolateral spinal fusion (PLF) is a procedure used for the treatment of degenerative spine disease. In this study we evaluated Osteogrow-C, a novel osteoinductive device comprised of recombinant human Bone morphogenetic protein 6 (rhBMP6) dispersed in autologous blood coagulum with synthetic ceramic particles, in the sheep PLF model. Osteogrow-C implants containing 74-420 or 1000-1700 µm ceramic particles (TCP/HA 80/20) were implanted between L4-L5 transverse processes in sheep (Ovis Aries, Merinolaandschaf breed). In the first experiment (n = 9 sheep; rhBMP6 dose 800 µg) the follow-up period was 27 weeks while in the second experiment (n = 12 sheep; rhBMP6 dose 500 µg) spinal fusion was assessed by in vivo CT after 9 weeks and at the end of the experiment after 14 (n = 6 sheep) and 40 (n = 6 sheep) weeks. Methods of evaluation included microCT, histological analyses and biomechanical testing. Osteogrow-C implants containing both 74-420 and 1000-1700 µm ceramic particles induced radiographic solid fusion 9 weeks following implantation. Ex-vivo microCT and histological analyses revealed complete osseointegration of newly formed bone with adjacent transverse processes. Biomechanical testing confirmed that fusion between transverse processes was complete and successful. Osteogrow-C implants induced spinal fusion in sheep PLF model and therefore represent a novel therapeutic solution for patients with degenerative disc disease.

Is canine calprotectin in serum stabile after storage at low temperature?

BACKGROUND: In human and veterinary medicine calprotectin is most widely used in diagnosing different gastro-intestinal diseases. The aim of this study was to assess the stability of canine calprotectin (cCP) in serum after storage at low temperatures and imprecision of the method. METHODS: Blood samples were collected from dogs with different clinical diagnoses. Twenty-two dogs were included in this study. Calprotectin concentration was measured 4 hours after serum separation (T0), and after being frozen at - 80 °C for 8 (T1) and 16 weeks (T2). The maximum permissible difference (MPD) was derived from the equation for calculating total error (TE) TE = %Bias + (1.96 x %CV), where bias and coefficient of variation (CV) were defined by the manufacturer. The dogs enrolled in this study were patients admitted during the morning (9-12 a.m.), on the day the first measurement was performed. All sample analysis for determination of stability were done in duplicates. For determination of within-run precision, the two patients' serum samples were analyzed in 20 replicates. Imprecision was assessed by analyzing 20 replicates on one plate on two samples where high and low concentrations were anticipated. RESULTS: The calculated value of MPD was 32.52%. Median calprotectin concentrations were higher at T1 114.08 µg/L (IQR = 55.05-254.56) and T2 133.6 µg/L (IQR = 100.57-332.98) than at T0 83.60 µg/L (IQR = 50.38-176.07). Relative and absolute bias at T1 (49.3%; 45.98 µg/L) and T2 (109.93%; 94.09 µg /L) have shown that calprotectin concentrations increase after long term storage at - 80 °C. CONCLUSION: The results of the present study indicate that c-CP was not stable for 16 weeks at low storage temperature (- 80 °C). Considering the observed change in the concentration of c-CP at T1, a storage time of 8 weeks should be safely applied. The method imprecision was not satisfactory, especially in the lower concentration range.

Changes in Acute Phase Response Biomarkers in Racing Endurance Horses.

This study aimed to evaluate if exercise-induced acute phase response (APR) occurs in endurance horses in response to the race. The study included 23 horses competing in an endurance competition with a successfully passed clinical examination before the race. Blood samples were collected before the start and within 30 min after the end of the race. Haematological and biochemical tests were performed and correlated to acute phase biomarkers changes. Values of calprotectin and haptoglobin (Hp) decreased after the races compared to values before, while concentrations of ceruloplasmin and albumin recorded a significant increase. Greater changes in calprotectin values were noted in Arabian horses compared to other breeds. Values of Hp showed a significantly greater decrease after longer races. Based on study results, endurance racing induces APR in horses characterised by significant changes in selected acute phase biomarkers. More pronounced changes were noted at races with higher average speeds, suggesting the need for thorough horse monitoring during exhausting races.

Nasal Chondrocyte-Based Engineered Grafts for the Repair of Articular Cartilage "Kissing" Lesions: A Pilot Large-Animal Study.

BACKGROUND: Bipolar or "kissing" cartilage lesions formed on 2 opposite articular surfaces of the knee joint are commonly listed as exclusion criteria for advanced cartilage therapies. PURPOSE: To test, in a pilot large-animal study, whether autologous nasal chondrocyte (NC)-based tissue engineering, recently introduced for the treatment of focal cartilage injuries, could provide a solution for challenging kissing lesions. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral kissing lesions were freshly introduced into the knee joints of 26 sheep and covered with NC-based grafts with a low or high hyaline-like extracellular matrix; a control group was treated with a cell-free scaffold collagen membrane (SCA). The cartilage repair site was assessed at 6 weeks and 6 months after implantation by histology, immunohistochemistry, and magnetic resonance imaging evaluation. RESULTS: NC-based grafts, independently of their composition, induced partial hyaline cartilage repair with stable integrity in surrounding healthy tissue at 6 months after treatment. The SCA repaired cartilage to a similar degree to that of NC-based grafts. CONCLUSION: Kissing lesion repair, as evidenced in this sheep study, demonstrated the feasibility of the treatment of complex cartilage injuries with advanced biological methods. However, the potential advantages of an NC-based approach over a cell-free approach warrant further investigations in a more relevant preclinical model. CLINICAL RELEVANCE: NC-based grafts currently undergoing phase II clinical trials have a high potential to replace existing cartilage therapies that show significant limitations in the quality and reproducibility of the repair method. We have brought this innovative concept to the next level by addressing a new clinical indication.

Evaluation of synthetic ceramics as compression resistant matrix to promote osteogenesis of autologous blood coagulum containing recombinant human bone morphogenetic protein 6 in rabbit posterolateral lumbar fusion model.

Posterolateral lumbar fusion (PLF) is a commonly performed surgical procedure for the treatment of pathological conditions of the lumbosacral spine. In the present study, we evaluated an autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) and synthetic ceramics used as compression resistant matrix (CRM) in the rabbit PLF model. In the pilot PLF rabbit experiment, we tested four different CRMs (BCP 500-1700 µm, BCP 1700-2500 µm and two different TCP in the form of slabs) which were selected based on achieving uniform ABC distribution. Next, ABGS implants composed of 2.5 mL ABC with 0.5 g ceramic particles (TCP or BCP (TCP/HA 80/20) of particle size 500-1700 µm) and 125 µg rhBMP6 (added to blood or lyophilized on ceramics) were placed bilaterally between transverse processes of the lumbar vertebrae (L5-L6) following exposition and decortication in 12 New Zealand White Rabbits observed for 7 weeks following surgery. Spinal fusion outcome was analysed by µCT, palpatory segmental mobility testing and selected specimens were either tested biomechanically (three-point bending test) and/or processed histologically. The total fusion success rate was 90.9% by both µCT analyses and by palpatory segmental mobility testing. The volume of newly formed bone between experimental groups with TCP or BCP ceramics and the different method of rhBMP6 application was comparable. The newly formed bone and ceramic particles integrated with the transverse processes on histological sections resulting in superior biomechanical properties. The results were retrospectively found superior to allograft devitalized mineralized bone as a CRM as reported previously in rabbit PLF. Overall, this novel ABGS containing rhBMP6, ABC and the specific 500-1700 µm synthetic ceramic particles supported new bone formation for the first time and successfully promoted posterolateral lumbar fusion in rabbits.

Autologous blood coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep.

In the present study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the L4-L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 µg rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) can serve as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion.

Metal projectile injuries in cats: review of 65 cases (2012-2014).

OBJECTIVES: The objective of this study was to compare the prevalence of different types of injuries caused by various types of projectiles among urban, suburban and rural cats of different ages in order to predict the type of injury sustained. METHODS: For the period 1 January 2012 to 30 April 2014, the medical records of cats with metal projectile injuries (PIs) were searched from the archive of the university's small animal diagnostic imaging centre. Age, sex, owner address, admission during a weekend or on a working day, month of admission, projectile position (head and neck; thoracic region; abdominal region, including lumbosacral spine; forelimbs; and hindlimbs, including tail), number of projectiles, presence of a wound and fracture related to the projectile, and the type of projectile were recorded for each cat. RESULTS: Sixty-five cats with PIs were admitted during the defined period. In 38.5% of cats the projectiles found upon radiography were incidental findings. The frequency of PIs peaked in March. Airgun projectiles were found in 80.0% of the cats. PIs in two or more body regions were found in 29.2% of the cats. Among the cats that had only been shot in one body region, the projectile was most frequently found in the abdominal region, including the lumbosacral spine (41.3%). CONCLUSIONS AND RELEVANCE: These results might prompt clinicians to evaluate closely and screen for feline PIs in emergency situations.

Review of 166 Gunshot Injury Cases in Dogs.

The study is aimed to establish predilection signalment and history data, and to investigate clinical findings and risk factors associated with a poor outcome in dogs with projectile injuries. A retrospective study was undertaken of 166 canine cases in which a projectile was found on radiograph in a university׳s diagnostic imaging center more than a 4-year period. The study included dogs with both apparent (obvious recent traumatic event) and incidental (traumatic event unknown to the owner) projectile injury. Radiographs were reviewed and data regarding projectile position according to body region, number and type of projectile(s), bone fracture(s), and wound(s) related to projectile were recorded. The dogs were divided into groups according to owner address, hunting accident vs. shooting unrelated to hunting, and projectile type found on radiographs. Overall, 160 dogs met the inclusion criteria, making 0.76% the incidence of gunshot injuries. Further, 91 dogs were received with incidental projectile injury, and 75 dogs had apparent projectile injury. Male dogs were overrepresented (74.1%). Hunting accidents were the cause of projectile injury in 12.7% of cases. Fractures were observed in 20.5% of dogs. Most of the dogs (62%) were from an urban area, and the most common projectile type was airgun projectile (62%). The risk of fatal outcome was 14.4 times higher in dogs with thoracic injuries. Projectile injuries are still a real cause of trauma, especially in urban areas and in male dogs. Most gunshot injuries do not cause fatalities, although the thoracic projectile injury was associated with a greater fatality risk.

Regional intravenous gentamicin administration for treatment of postoperative tarso-metatarsal infection in a dog--a case report.

The regional intravenous antibiotic administration has been used to achieve high local concentrations of antibiotics into the extremities. The goal of this case report was to describe a known, but not often used technique of local administration of antibiotics for treatment of acute soft tissue, joint or bone infection in a dog, which suffered from chronic renal failure. In a seven years old Great Dane tarso-metatarsal arthrodesis was performed and three days after surgery infection was obvious. Gentamicin-sensitive E. co i was found by antibiogram. A tourniquet was tightened on the affected limb and gentamicin was administered in a dosage of 0,5 mg/kg every twelve hours via an intravenous catheter placed in the saphenous vein. The therapy lasted for ten days and wound healing was progressive. Regional intravenous gentamicin administration was very effective in treating distal extremity infection.

Bone morphogenetic protein (BMP)1-3 enhances bone repair.

Members of the astacin family of metalloproteinases such as human bone morphogenetic protein 1 (BMP-1) regulate morphogenesis by processing precursors to mature functional extracellular matrix (ECM) proteins and several growth factors including TGFß, BMP2, BMP4 and GFD8. We have recently discovered that BMP1-3 isoform of the Bmp-1 gene circulates in the human plasma and is significantly increased in patients with acute bone fracture. We hypothesized that circulating BMP1-3 might have an important role in bone repair and serve as a novel bone biomarker. When administered systemically to rats with a long bone fracture and locally to rabbits with a critical size defect of the ulna, recombinant human BMP1-3 enhanced bone healing. In contrast, neutralization of the endogenous BMP1-3 by a specific polyclonal antibody delayed the bone union. Invitro BMP1-3 increased the expression of collagen type I and osteocalcin in MC3T3-E(1) osteoblast like cells, and enhanced the formation of mineralized bone nodules from bone marrow mesenchymal stem cells. We suggest that BMP1-3 is a novel systemic regulator of bone repair.

[Histologic assessment of tissue healing of hyaline cartilage by use of semiquantitative evaluation scale]. / Histoloska procjena cijeljenja zglobne hrskavice upotrebom semikvantitativne ocjenske ljestvice.

INTRODUCTION: Articular cartilage is an avascular and aneural tissue lacking lymph drainage, hence its inability of spontaneous repair following injury. Thus, it offers an interesting model for scientific research. A number of methods have been suggested to enhance cartilage repair, but none has yet produced significant success. The possible application of the aforementioned methods has brought about the necessity to evaluate their results. The objective of this study was to analyze results of a study of the effects of the use of TGF-beta gene transduced bone marrow clot on articular cartilage defects using ICRS visual histological assessment scale. METHODS: The research was conducted on 28 skeletally mature sheep that were randomly assigned to four groups and surgically inflicted femoral chondral defects. The articular surfaces were then treated with TGF-beta1 gene transduced bone marrow clot (TGF group), GFP transduced bone marrow clot (GFP group), untransduced bone marrow clot (BM group) or left untreated (NC group). The analysis was performed by visual examination of cartilage samples and results were obtained using ICRS visual histological assessment scale. The results were subsequently subjected to statistical assessment using Kruskal-Wallis and Mann-Whitney tests. RESULTS: Kruskal-Wallis test yielded statistically significant difference with respect to cell distribution. Mann-Whitney test showed statistically significant difference between TGF and NC groups (P = 0.002), as well as between BM and NC groups (P = 0.002 with Bonferroni correction). DISCUSSION: Twenty-six of the twenty-eight samples were subjected to histologic and subsequent statistical analysis; two were discarded due to faulty histology technique. Our results indicated a level of certainty as to the positive effect of TGF-beta1 gene transduced bone marrow clot in restoration of articular cartilage defects. However, additional research is necessary in the field. One of the significant drawbacks on histologic assessment of cartilage samples were the errors in histologic preparation, for which some samples had to be discarded and significantly impaired the analytical quality of the others. Defects of structures surrounding the articular cartilage, e.g., subchondral bone or connective tissue, might also impair the quality of histologic analysis. Additional analyses, i.e. polarizing microscopy should be performed to determine the degree of integration of the newly formed tissue with the surrounding cartilage. The semiquantitative ICRS scale, although of great practical value, has limitations as to the objectivity of the assessment, taking into account the analytical ability of the evaluator, as well as the accuracy of semiquantitative analysis in comparison to the methods of quantitative analysis. CONCLUSION: Overall results of histologic analysis indicated that the application of TGF-beta1 gene transduced bone marrow clot could have measurable clinical effects on articular cartilage repair. The ICRS visual histological assessment scale is a valuable analytical method for cartilage repair evaluation. In this respect, further analyses of the method value would be of great importance.

Cardiopulmonary effects of hemorrhagic shock in splenic autotransplanted pigs: a new surgical model.

The spleen is an important organ for hemodynamic compensation during hemorrhagic shock. The aim of the study was to compare the hemodynamic and metabolic responses of sham-operated pigs with intact spleen, splenectomized pigs, and splenic autotransplanted pigs during hemorrhagic shock. Hemorrhagic shock was induced by 30% total blood volume bleed in sham-operated, splenectomized and splenic autotransplanted pigs (n = 20). Cardiopulmonary and metabolic variables were measured before, immediately after, and at 20, 60 and 100 minutes after hemorrhage. Upon hemorrhagic shock induction, body temperature, mean arterial pressure, mean pulmonary arterial pressure, cardiac output, cardiac index and oxygen delivery decreased, while lactate and shock index increased. Hemoglobin and hematocrit were significantly lower in the splenectomized and splenic autotransplant groups as compared with the control group at 60 and 100 minutes after hemorrhage (p < 0.05). Unlike intact spleen, splenic autotransplant could not improve hemodynamic parameters in hemorrhagic shock in pigs. In comparison to mice, rats or dogs, this species could be an interesting investigation model to test new surgical procedures during splenic related hemorrhagic shock, with potential applications in human medicine.

Effect of splenectomy and autologous spleen transplantation on the serum platelet-activating factor acetylhydrolase (PAF-AH) activity and acute phase response (APR) in a porcine model.

The aim of this study was to evaluate the inflammatory response after total splenectomy and spleen autotransplantation in a porcine model by measuring serum platelet-activating factor acetylhydrolase activity, C-reactive protein and albumin concentrations. Nineteen piglets were used in the experiment. After induction of anesthesia, animals were randomly divided into three groups: sham-operation with spleens intact (n = 6), total splenectomy (n = 6), and splenic autotransplantation (n = 7) with small fragments of the spleen autotransplanted into the greater omentum. The blood samples were taken just before surgery and on day 1st, 5th, 12th, 26th and 40th postoperatively. PAF-AH activity, CRP and albumin concentrations were assayed in the sera. After total splenectomy, PAF-AH activity was significantly increased on day 5th, while there was no significant increase after spleen autotransplantation or the sham-operation. CRP was significantly increased after surgery in all experimental groups. Albumin was significantly decreased after surgery from day 5th until day 40th in splenectomized and autotransplanted pigs. Increased PAF-AH activity after splenectomy and spleen autotransplantation might be attributed to inflammatory conditions due to the loss of splenic tissue and trauma. Time-course increase of CRP, in all groups after surgery suggests post-injury inflammatory response due to tissue lesion during operation.