Stewart D. Ryan

In situ handheld three-dimensional bioprinting for cartilage regeneration

Articular cartilage injuries experienced at an early age can lead to the development of osteoarthritis later in life. In situ three‐dimensional (3D) printing is an exciting and innovative biofabrication technology that enables the surgeon to deliver tissue‐engineering techniques at the time and location of need. We have created a hand‐held 3D printing device (biopen) that allows the simultaneous coaxial extrusion of bioscaffold and cultured cells directly into the cartilage defect in vivo in a single‐session surgery. This pilot study assessed the ability of the biopen to repair a full‐thickness chondral defect and the early outcomes in cartilage regeneration, and compared these results with other treatments in a large animal model. A standardized critical‐sized full‐thickness chondral defect was created in the weight‐bearing surface of the lateral and medial condyles of both femurs of six sheep. Each defect was treated with one of the following treatments: (i) hand‐held in situ 3D printed bioscaffold using the biopen (HH group), (ii) preconstructed bench‐based printed bioscaffolds (BB group), (iii) microfractures (MF group) or (iv) untreated (control, C group). At 8 weeks after surgery, macroscopic, microscopic and biomechanical tests were performed. Surgical 3D bioprinting was performed in all animals without any intra‐ or postoperative complication. The HH biopen allowed early cartilage regeneration. The results of this study show that real‐time, in vivo bioprinting with cells and scaffold is a feasible means of delivering a regenerative medicine strategy in a large animal model to regenerate articular cartilage.

Outcomes of Limb-Sparing Surgery Using Two Generations of Metal Endoprosthesis in 45 Dogs With Distal Radial Osteosarcoma. A Veterinary Society of Surgical Oncology Retrospective Study

OBJECTIVE To report outcomes in dogs with distal radial osteosarcoma (OSA) treated with metal endoprosthesis limb-sparing surgery and compare outcomes between 2 generations of endoprosthesis. STUDY DESIGN Multi-institutional retrospective case series. ANIMALS Forty-five dogs with distal radial OSA treated with endoprosthesis and chemotherapy. METHODS Data of dogs treated with either first-generation endoprosthesis (GEN1) or second-generation endoprosthesis (GEN2) were sourced from medical records and radiographs. Surgical outcomes included postoperative lameness assessment and the presence, severity, and time to onset of complications. Oncologic outcomes included presence of local recurrence or metastasis, time to onset of local recurrence, metastasis-free interval (MFI), and survival time. Results for surgical and oncologic outcomes were compared between GEN1 and GEN2. RESULTS Twenty-eight dogs received GEN1 and 17 dogs received GEN2. There were 39 complications (96%, 14 minor, 29 major) including infection (78%), implant-related complication (36%), and local recurrence (24%). Metastatic frequency was 67% and median MFI was 188 days (95% confidence interval [CI]: 126-250 days). Survival time ranged from 34 days to 6.1 years with a median of 289 days (95% CI: 207-371 days). There was no significant difference in complication severity, frequency, time to complication, MFI, or survival time between dogs receiving GEN1 and GEN2. CONCLUSION There was no significant difference in outcomes between dogs receiving GEN1 and GEN2 for limb-sparing surgery of the radius. Metastatic frequency and survival time for metal endoprosthesis were similar to that of amputation with curative intent chemotherapy.

Determination of mechanical properties of canine carpal ligaments.

OBJECTIVE To evaluate the mechanical properties of canine carpal ligaments for use in a finite element model of the canine antebrachium. SAMPLE POPULATION 26 forelimbs obtained from cadavers of 13 dogs euthanized for reasons unrelated to this study. PROCEDURES 6 ligaments (medial collateral, lateral collateral, palmar ulnocarpal, palmar radiocarpal, accessorometacarpal-V, and accessorometacarpal-IV) were evaluated. Quasistatic tensile tests were performed on all specimens (n = 8 specimens/ligament) by use of a servohydraulic materials testing system in conjunction with a 6-df load cell. Each specimen was preconditioned for 10 cycles by applying 2% strain by use of a Haversine waveform. Tension was subsequently applied to each specimen at a strain rate of 0.5%/s until ligament failure. RESULTS Significant differences in modulus of elasticity were detected among the ligaments. Elastic modulus did not differ significantly between the 2 accessorometacapal ligaments, between the 2 collateral ligaments, or between the 2 palmar carpal ligaments. Ligaments were classified into 3 groups (accessorometacarpal ligaments, intra-articular ligaments, and palmar carpal ligaments), and significant differences were detected among the 3 ligament groups. The accessorometacarpal ligaments had a relatively high elastic modulus, compared with results for the other ligaments. The medial and lateral collateral ligaments had the lowest elastic modulus of any of the ligaments tested. CONCLUSIONS AND CLINICAL RELEVANCE These results indicated a strong function-elastic modulus relationship for the 6 ligaments tested. The mechanical properties described here will be of use in creating a finite element model of the canine antebrachium.

Comparison of Alternate and Simultaneous Tensioning of Wires in a Single‐Ring Fixator Construct

OBJECTIVE To measure and compare the strain of wires tensioned with alternate (ALT) and simultaneous (SIM) tensioning in a single-ring fixator construct and compare the stiffness of these constructs under axial loading. STUDY DESIGN Experimental mechanical study. SAMPLE POPULATION Twenty-four, 84 mm diameter, single-ring constructs. METHODS Twenty-four, 84 mm diameter, single-ring constructs were assembled using 2 1.6 mm wires placed at a 60 degrees angle tensioned with either ALT or SIM technique to 90 kg tension. Voltage data from a strain gauge were recorded during the wire-tensioning process, cyclic axial loading, and load-to-failure testing. Wire strains were calculated for each wire and compared within constructs and between ALT and SIM groups. Construct stiffness was compared between groups. RESULTS There was no difference between the tensioning methods in final wire strains after initial tensioning for both the wire below the ring (W1; P=.698) and the wire above the ring (W2; P=.233). There was also no difference in final wire strains within each tensioning method group (ALT, P=.289; SIM, P=.583). Loss of wire strain (3.5-5%) occurred after cyclic loading for both wires in both groups. There was no difference in construct stiffness between the ALT and SIM groups (P=.126). Mode of failure was by wire breakage in all constructs and occurred most frequently in W1. CONCLUSION ALT tensioning of wires produced similar wire strains within a single-ring construct after initial tensioning to SIM tensioned wires. There was no difference in construct stiffness under axial loading between AIM and SIM tensioned constructs. CLINICAL RELEVANCE ALT tensioning of wires in a single-ring fixator construct can be used as an alternative to SIM tensioning, as similar initial wire tensions are achieved.

Clinical outcome of 42 dogs with scapular tumors treated by scapulectomy: a Veterinary Society of Surgical Oncology (VSSO) retrospective study (1995–2010)

OBJECTIVE To report signalment, clinical signs, preoperative staging tests, histologic diagnosis, surgical, and oncologic outcomes including postoperative limb use, in dogs with scapular tumors treated by scapulectomy. STUDY DESIGN Retrospective case series ANIMALS Dogs (n = 42) with scapular tumors. METHODS Medical records (1995-2010) from 6 hospitals were searched for dogs with scapular tumors treated by scapulectomy. Data retrieved were: signalment, weight, percentage of scapula removed, histologic diagnosis, postoperative limb use, adjunctive therapy, disease free interval (DFI), and survival time (ST). Individual variables were modeled with a Cox proportional hazard model accounting for censoring to determine risk factors for decreased DFI and ST. For categorical variables, Kaplan-Meier survival plots as well as mean and median survival times (MSTs) were calculated. RESULTS Subtotal scapulectomy was performed in 18 dogs (42.9%). Osteosarcoma (OSA) was diagnosed in 27 dogs (64.3%). Limb use was evaluated immediately after surgery in 41 dogs. Information on limb use at other times (1, 2, 3, and >3 months) postoperatively was also available for some dogs and was good to excellent overall. Only adjunctive chemotherapy had a positive significant effect on DFI (P = .00011) and ST (P = .0003). CONCLUSION Canine scapular tumors can be treated effectively by scapulectomy and limb use is fair to excellent for most dogs. OSA was the most common scapular tumor. Overall prognosis for scapular OSA is similar to appendicular OSA at other sites and use of adjunctive chemotherapy prolonged the overall DFI and MST.

Orthotopic model of canine osteosarcoma in athymic rats for evaluation of stereotactic radiotherapy

OBJECTIVE To develop an orthotopic model of canine osteosarcoma in athymic rats as a model for evaluating the effects of stereotactic radiotherapy (SRT) on osteosarcoma cells. ANIMALS 26 athymic nude rats. PROCEDURES 3 experiments were performed. In the first 2 experiments, rats were injected with 1 × 10(6) Abrams canine osteosarcoma cells into the proximal aspect of the tibia (n = 12) or distal aspect of the femur (6). Tumor engraftment and progression were monitored weekly via radiography, luciferase imaging, and measurement of urine pyridinoline concentration for 5 weeks and histologic evaluation after euthanasia. In the third experiment, 8 rats underwent canine osteosarcoma cell injection into the distal aspect of the femur and SRT was administered to the affected area in three 12-Gy fractions delivered on consecutive days (total radiation dose, 36 Gy). Percentage tumor necrosis and urinary pyridinoline concentrations were used to assess local tumor control. The short-term effect of SRT on skin was also evaluated. RESULTS Tumors developed in 10 of 12 tibial sites and all 14 femoral sites. Administration of SRT to rats with femoral osteosarcoma was feasible and successful. Mean tumor necrosis of 95% was achieved histologically, and minimal adverse skin effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE The orthotopic model of canine osteosarcoma in rats developed in this study was suitable for evaluating the effects of local tumor control and can be used in future studies to evaluate optimization of SRT duration, dose, and fractionation schemes. The model could also allow evaluation of other treatments in combination with SRT, such as chemotherapy or bisphosphonate, radioprotectant, or parathyroid hormone treatment.

Characterization of differences in calculated and actual measured skin doses to canine limbs during stereotactic radiosurgery using Gafchromic film

Accurate calculation of absorbed dose to the skin, especially the superficial and radiosensitive basal cell layer, is difficult for many reasons including, but not limited to, the build-up effect of megavoltage photons, tangential beam effects, mixed energy scatter from support devices, and dose interpolation caused by a finite resolution calculation matrix. Stereotactic body radiotherapy (SBRT) has been developed as an alternative limb salvage treatment option at Colorado State University Veterinary Teaching Hospital for dogs with extremity bone tumors. Optimal dose delivery to the tumor during SBRT treatment can be limited by uncertainty in skin dose calculation. The aim of this study was to characterize the difference between measured and calculated radiation dose by the Varian Eclipse (Varian Medical Systems, Palo Alto, CA) AAA treatment planning algorithm (for 1-mm, 2-mm, and 5-mm calculation voxel dimensions) as a function of distance from the skin surface. The study used Gafchromic EBT film (International Specialty Products, Wayne, NJ), FilmQA analysis software, a limb phantom constructed from plastic water™ (fluke Biomedical, Everett, WA) and a canine cadaver forelimb. The limb phantom was exposed to 6-MV treatments consisting of a single-beam, a pair of parallel opposed beams, and a 7-beam coplanar treatment plan. The canine forelimb was exposed to the 7-beam coplanar plan. Radiation dose to the forelimb skin at the surface and at depths of 1.65 mm and 1.35 mm below the skin surface were also measured with the Gafchromic film. The calculation algorithm estimated the dose well at depths beyond buildup for all calculation voxel sizes. The calculation algorithm underestimated the dose in portions of the buildup region of tissue for all comparisons, with the most significant differences observed in the 5-mm calculation voxel and the least difference in the 1-mm voxel. Results indicate a significant difference between measured and calculated data extending to average depths of 2.5 mm, 3.4 mm, and 10 mm for the 1-mm, 2-mm, and 5-mm dimension calculation matrices, respectively. These results emphasize the importance of selecting as small a treatment planning software calculation matrix dimension as is practically possible and of taking a conservative approach for skin treatment planning objectives. One suggested conservative approach is accomplished by defining the skin organ as the outermost 2-3 mm of the body such that the high dose tail of the skin organ dose-volume histogram curve represents dose on the deep side of the skin where the algorithm is more accurate.

Osteosarcoma following tibial plateau leveling osteotomy in dogs: 29 cases (1997–2011)

OBJECTIVE To determine the signalment, tibial plateau leveling osteotomy (TPLO) plate type, clinical staging information, treatment, and oncological outcome in dogs that developed osteosarcoma at the proximal aspect of the tibia following TPLO and to calculate the interval between TPLO and osteosarcoma diagnosis. DESIGN Multi-institutional retrospective case series. ANIMALS 29 dogs. PROCEDURES Medical records from 8 participating institutions were searched for dogs that developed osteosarcoma (confirmed through cytologic or histologic evaluation) at previous TPLO sites. Signalment, TPLO details, staging tests, treatment data, and outcome information were recorded. Descriptive statistics were calculated, and disease-free intervals and survival times were evaluated by means of Kaplan-Meier analysis. RESULTS 29 dogs met the inclusion criteria. The mean age was 9.2 years and mean weight was 45.1 kg (99.2 lb) at the time of osteosarcoma diagnosis. Most dogs had swelling over the proximal aspect of the tibia (17/21) and lameness of the affected limb (28/29). The mean interval between TPLO and osteosarcoma diagnosis was 5.3 years. One type of cast stainless steel TPLO plate was used in most (18) dogs; the remaining dogs had received plates of wrought stainless steel (n = 4) or unrecorded type (7). Twenty-three of 29 dogs underwent treatment for osteosarcoma. Median survival time for 10 dogs that underwent amputation of the affected limb and received ≥ 1 chemotherapeutic treatment was 313 days. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that osteosarcoma should be a differential diagnosis for dogs with a history of TPLO that later develop lameness and swelling at the previous surgical site. Oncological outcome following amputation and chemotherapy appeared to be similar to outcomes previously reported for dogs with appendicular osteosarcoma.

Immunohistochemical Validation of Spontaneously Arising Canine Osteosarcoma as a Model for Human Osteosarcoma

Osteosarcoma (OS) originates from bone-forming mesenchymal cells and represents one of the primary bone tumours. It is the most common primary bone tumour in dogs and man. The characterization of an appropriate natural disease animal model to study human OS is essential to elucidate the pathogenesis of the disease. This study aimed to validate canine OS as a model for the human disease by evaluating immunohistochemically the expression of markers known to be important in human OS. The immunohistochemical panel included vimentin, alkaline phosphatase (ALP), desmin, S100, neuron-specific enolase (NSE), runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 4 (BMP4). Immunohistochemistry was conducted on formalin-fixed, paraffin wax-embedded tissue sections from 59 dogs with confirmed primary OS. Vimentin, ALP, Runx2 and BMP4 were highly expressed by all tumours, while desmin, S100 and NSE were expressed variably. The findings were similar to those described previously for human OS and suggest that canine OS may represent a useful model for the study of the human disease.

Bi-institutional retrospective cohort study evaluating the incidence of osteosarcoma following tibial plateau levelling osteotomy (2000–2009)

OBJECTIVES To evaluate the incidence and risk factors for occurrence of osteosarcoma (OSA) following tibial plateau levelling osteotomy (TPLO). METHODS Medical records of client-owned dogs that underwent consecutive TPLO procedures at two institutions were retrospectively reviewed. Referring veterinarians and owners were contacted for follow-up. Each institutional cohort was assessed separately, and the incidence density rate and median time to occurrence of OSA at the TPLO site and at other sites were calculated. Marginal Cox regression was used to calculate hazard ratios and 95% confidence intervals for potential risk factors for occurrence of OSA. RESULTS There were 472 CLINIC A (Colorado State University Veterinary Teaching Hospital) and 1992 CLINIC B (SAGE Centers for Veterinary Specialty and Emergency Care) TPLO cases with over one year of follow-up available. There were five and six dogs within the cohorts that developed OSA at the site of TPLO, and seven and 22 dogs that developed OSA at other anatomical sites, respectively. The incidence density rates of OSA at the TPLO site were 30.4 and 10.2 per 10,000 dog-years at risk, and other sites were 42.6 and 37.5 per 10,000 dog-years at risk. The median time to occurrence of OSA of TPLO site OSA was 4.6 and 4.4 years, which was longer than that of other site OSA of 2.9 and 3.4 years. CLINICAL SIGNIFICANCE There is a low incidence of OSA following TPLO surgery. The longer time to occurrence for TPLO site OSA is similar to that for fracture-associated sarcoma, and could indicate a similar underlying pathophysiology rather than spontaneous OSA occurrence.