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Gelatin sponge enhances the efficiency and localization of adenovirus mediated gene transfer in healing rat achilles tendon
Gelatin sponge e n h a n c e s the e f f i c i e n c y and localization of adenovirus m e d i a t e d g en e t ransf er in healing rat achilles tendon Q. Dai*, L.Manfield,Y. Wang& G. Murrell Orthopaedic Research Institute
Adenovirus-mediated gene transfer is a potential method for enhancing tendon healing. We investigated feasibJlity of the gene transfer to healing rat Achilles tendon, developed a novel method to enhance transfection efficiency by using a gelatin sponge. We used replication deficient adenovirus, Ad5CMVntLacZ, containing the reporter Escherichia coil 13-galactosidase; along with Ad5CMVempty, adenoviral vector without inserted gene, as control. The healing tendon model consisted of a 3 mm circular defect made four days after surgical division of the tendon. Adenoviruses were injected into the hole, or soaked in a gelatin sponge and that sponge implanted into the hole. Transfection efficiency was determined by X-gal staining of tissue samples and frozen sections of tissue. Successful transfection was obtained as confirmed by X-gal s!aining. 0.4% of tendon cells were transfected when 106 PFU Ad5CMVntLacZ was injected. The rate rose to 2% with 108 PFU and 3% with 10g PFU. The duration of 13galactosJd~se expression was 17 days. Transfection efficiency was enhanced threefold and Iocalizatiod improved when gelatin sponge was used. Adenovirus can transfer a gene of interest to healing rat tendon without adverse effects. Gelatin sponge implantation can enhance the efficiency and localization of adenoviral transfection in vivo.
T e n o c y t e morphology c h a n g e s o c c u r before ground s u b s t a n c e , collagen or v a s c u l a r c h a n g e s in t e n d i n o p a t h y J. Cook .1, F.Bonar 2, J. Feller 1, S. Kiss 3 & K. Khan 4 1Musculoskeletal Research Centre 2Douglass-Hanly-Moir Pathology 3Radclin Imaging 4University of British Columbia
The early stages of the pathology process in overuse tendinopathy has been hypothesized, but not shown experimentally. We examined the patellar tendons of asymptomatic, athletes with normal ultrasound to see if the process of tendon disease in the early stages could be identified. Athletes prior to patellar tendon knee reconstruction completed a questionnaire detailing history of symptoms and activity levels and had an ultrasound examination of their patellar tendon. At surgery, a tissue sample was taken from the posterocentral part of the graft. The tissue was fixed, stained and examined under light microscopy. Tendons were evaluated for changes in tenocyte appearance, ground substance stainability, collagen fibre separation and increased vascularity. Histopathology was normal in 14 tendons, 12 tendons were abnormal in some or most of the histopathology. No tendons had neovascularisation, a hallmark of overuse tendinopathy. There was no difference in hours/week of activity between those with normal tendons and those with pathology. There were three tendons that showed changes in cellular activation without any other tendon changes. Three tendons that had grOund substance increase showed tenocyte activation, similarly five tendons that had collagen separation always had changes in ground substance and tenocyte activation. These findings suggest the process of tendinopathy maybe a cascade of cellular change, ground substance increase, collagen separation, vascularity increase. Further research is needed to confirm these findings.
Adenovirus-mediated gene transfer is a potential method for enhancing tendon healing. We investigated feasibJlity of the gene transfer to healing rat Achilles tendon, developed a novel method to enhance transfection efficiency by using a gelatin sponge. We used replication deficient adenovirus, Ad5CMVntLacZ, containing the reporter Escherichia coil 13-galactosidase; along with Ad5CMVempty, adenoviral vector without inserted gene, as control. The healing tendon model consisted of a 3 mm circular defect made four days after surgical division of the tendon. Adenoviruses were injected into the hole, or soaked in a gelatin sponge and that sponge implanted into the hole. Transfection efficiency was determined by X-gal staining of tissue samples and frozen sections of tissue. Successful transfection was obtained as confirmed by X-gal s!aining. 0.4% of tendon cells were transfected when 106 PFU Ad5CMVntLacZ was injected. The rate rose to 2% with 108 PFU and 3% with 10g PFU. The duration of 13galactosJd~se expression was 17 days. Transfection efficiency was enhanced threefold and Iocalizatiod improved when gelatin sponge was used. Adenovirus can transfer a gene of interest to healing rat tendon without adverse effects. Gelatin sponge implantation can enhance the efficiency and localization of adenoviral transfection in vivo.
T e n o c y t e morphology c h a n g e s o c c u r before ground s u b s t a n c e , collagen or v a s c u l a r c h a n g e s in t e n d i n o p a t h y J. Cook .1, F.Bonar 2, J. Feller 1, S. Kiss 3 & K. Khan 4 1Musculoskeletal Research Centre 2Douglass-Hanly-Moir Pathology 3Radclin Imaging 4University of British Columbia
The early stages of the pathology process in overuse tendinopathy has been hypothesized, but not shown experimentally. We examined the patellar tendons of asymptomatic, athletes with normal ultrasound to see if the process of tendon disease in the early stages could be identified. Athletes prior to patellar tendon knee reconstruction completed a questionnaire detailing history of symptoms and activity levels and had an ultrasound examination of their patellar tendon. At surgery, a tissue sample was taken from the posterocentral part of the graft. The tissue was fixed, stained and examined under light microscopy. Tendons were evaluated for changes in tenocyte appearance, ground substance stainability, collagen fibre separation and increased vascularity. Histopathology was normal in 14 tendons, 12 tendons were abnormal in some or most of the histopathology. No tendons had neovascularisation, a hallmark of overuse tendinopathy. There was no difference in hours/week of activity between those with normal tendons and those with pathology. There were three tendons that showed changes in cellular activation without any other tendon changes. Three tendons that had grOund substance increase showed tenocyte activation, similarly five tendons that had collagen separation always had changes in ground substance and tenocyte activation. These findings suggest the process of tendinopathy maybe a cascade of cellular change, ground substance increase, collagen separation, vascularity increase. Further research is needed to confirm these findings.