The objectives of this study were to determine how culture time and dynamic compression, applied ... more The objectives of this study were to determine how culture time and dynamic compression, applied to murine chondrocyte–agarose constructs, influence construct stiffness, expression of col2 and type II collagen. Chon-drocytes were harvested from the ribs of six newborn double transgenic mice carrying transgenes that use enhanced cyan fluorescent protein (ECFP) and green fluorescent protein (GFP-T) as reporters for expression from the col2a1 and col1a1 promoters, respectively. Sixty-three constructs (8 mm diameter 3 mm thick) per animal were created by seeding chondrocytes (10 106 per mL) in agarose gel (2 % w=v). Twenty-eight constructs from each animal were stimulated for 7, 14, 21, or 28 days in a custom bioreactor housed in an electromagnetic system. Twenty-eight constructs exposed to identical culture conditions but without mechanical stimulation served as nonstimulated controls for 7, 14, 21, and 28 days. The remaining seven constructs served as day 0 controls. Fluorescing cells...
Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered ... more Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n = 10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After ...
Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruci... more Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance du...
The objectives of this study were to determine how tensile stimulation delivered up to 14 days in... more The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 × 106 mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h/day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.
To cite this article: David L. Butler, Shawn A. Hunter, Kumar Chokalingam, Michael J. Cordray, Ja... more To cite this article: David L. Butler, Shawn A. Hunter, Kumar Chokalingam, Michael J. Cordray, Jason Shearn, Natalia Juncosa-Melvin, Sanjit Nirmalanandhan, Abhishek Jain. Tissue Engineering Part A. April 2009, 15(4): 741-749. doi:10.1089/ten.tea.2008.0292.
Over 16 million patients present each year in the US with tendon, ligament, and joint injuries at... more Over 16 million patients present each year in the US with tendon, ligament, and joint injuries at a cost of $30 billion per year [1]. To treat tendon injuries, our lab employs principles of functional tissue engineering (FTE) to first characterize normal in vivo tendon function during activities of daily living (ADLs) to then guide creation of cell-scaffold tissue engineered constructs (TECs) that meet these functional demands after surgery [2-5]. Much of our work has focused on repairing a central patellar tendon defect in the rabbit using mechanically-preconditioned TECs composed of mesenchymal progenitor cells (MPC) in collagen-based scaffolds. While these treatments have achieved many of our functional goals, further improvements require that we consider the tendon's biological design criteria in addition to the mechanical parameters. In this context, our strategy has been to understand how to mimic aspects of normal murine tendon development to better design TECs that impro...
Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2015
This study determined how anterior cruciate ligament (ACL) reconstruction affected the magnitude ... more This study determined how anterior cruciate ligament (ACL) reconstruction affected the magnitude and temporal patterns of anterior knee force and internal knee moment during 2000 cycles of simulated gait. Porcine knees were tested using a six degree-of-freedom robot, examining three porcine allograft materials compared with the native ACL. Reconstructions were performed using: (1) bone-patellar tendon-bone allograft (BPTB), (2) reconstructive porcine tissue matrix (RTM), or (3) an RTM-polymer hybrid construct (Hybrid). Forces and moments were measured over the entire gait cycle and contrasted at heel strike, mid stance, toe off, and peak flexion. The Hybrid construct performed the best, as magnitude and temporal changes in both anterior knee force and internal knee moment were not different from the native ACL knee. Conversely, the RTM knees showed greater loss in anterior knee force during 2000 cycles than the native ACL knee at heel strike and toe off, with an average force loss o...
The Journal of bone and joint surgery. American volume, 1984
Virtually all types of collagenous tissues have been transferred in and around the knee joint for... more Virtually all types of collagenous tissues have been transferred in and around the knee joint for intra-articular and extra-articular ligament reconstructions. However, the mechanical properties (in particular, strength) of such grafts have not been determined in tissues from young adult donors, where age and disuse-related effects have been excluded. To provide this information, we subjected ligament graft tissues to high-strain-rate failure tests to determine their strength and elongation properties. The results were compared with the mechanical properties of anterior cruciate ligaments from a similar young-adult donor population. The study indicated that some graft tissues used in ligament reconstructions are markedly weak and therefore are at risk for elongation and failure at low forces. Grafts utilizing prepatellar retinacular tissues (as in certain anterior-cruciate reconstructions) and others in which a somewhat narrow width of fascia lata or distal iliotibial tract is utili...
The objectives of this study were to determine how culture time and dynamic compression, applied ... more The objectives of this study were to determine how culture time and dynamic compression, applied to murine chondrocyte–agarose constructs, influence construct stiffness, expression of col2 and type II collagen. Chon-drocytes were harvested from the ribs of six newborn double transgenic mice carrying transgenes that use enhanced cyan fluorescent protein (ECFP) and green fluorescent protein (GFP-T) as reporters for expression from the col2a1 and col1a1 promoters, respectively. Sixty-three constructs (8 mm diameter 3 mm thick) per animal were created by seeding chondrocytes (10 106 per mL) in agarose gel (2 % w=v). Twenty-eight constructs from each animal were stimulated for 7, 14, 21, or 28 days in a custom bioreactor housed in an electromagnetic system. Twenty-eight constructs exposed to identical culture conditions but without mechanical stimulation served as nonstimulated controls for 7, 14, 21, and 28 days. The remaining seven constructs served as day 0 controls. Fluorescing cells...
Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered ... more Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n = 10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After ...
Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruci... more Activities of daily living (ADLs) generate complex, multidirectional forces in the anterior cruciate ligament (ACL). While calibration problems preclude direct measurement in patients, ACL forces can conceivably be measured in animals after technical challenges are overcome. For example, motion and force sensors can be implanted in the animal but investigators must determine the extent to which these sensors and surgery affect normal gait. Our objectives in this study were to determine (1) if surgically implanting knee motion sensors and an ACL force sensor significantly alter normal ovine gait and (2) how increasing gait speed and grade on a treadmill affect ovine gait before and after surgery. Ten skeletally mature, female sheep were used to test four hypotheses: (1) surgical implantation of sensors would significantly decrease average and peak vertical ground reaction forces (VGRFs) in the operated limb, (2) surgical implantation would significantly decrease single limb stance du...
The objectives of this study were to determine how tensile stimulation delivered up to 14 days in... more The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 × 106 mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h/day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary the mechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.
To cite this article: David L. Butler, Shawn A. Hunter, Kumar Chokalingam, Michael J. Cordray, Ja... more To cite this article: David L. Butler, Shawn A. Hunter, Kumar Chokalingam, Michael J. Cordray, Jason Shearn, Natalia Juncosa-Melvin, Sanjit Nirmalanandhan, Abhishek Jain. Tissue Engineering Part A. April 2009, 15(4): 741-749. doi:10.1089/ten.tea.2008.0292.
Over 16 million patients present each year in the US with tendon, ligament, and joint injuries at... more Over 16 million patients present each year in the US with tendon, ligament, and joint injuries at a cost of $30 billion per year [1]. To treat tendon injuries, our lab employs principles of functional tissue engineering (FTE) to first characterize normal in vivo tendon function during activities of daily living (ADLs) to then guide creation of cell-scaffold tissue engineered constructs (TECs) that meet these functional demands after surgery [2-5]. Much of our work has focused on repairing a central patellar tendon defect in the rabbit using mechanically-preconditioned TECs composed of mesenchymal progenitor cells (MPC) in collagen-based scaffolds. While these treatments have achieved many of our functional goals, further improvements require that we consider the tendon's biological design criteria in addition to the mechanical parameters. In this context, our strategy has been to understand how to mimic aspects of normal murine tendon development to better design TECs that impro...
Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2015
This study determined how anterior cruciate ligament (ACL) reconstruction affected the magnitude ... more This study determined how anterior cruciate ligament (ACL) reconstruction affected the magnitude and temporal patterns of anterior knee force and internal knee moment during 2000 cycles of simulated gait. Porcine knees were tested using a six degree-of-freedom robot, examining three porcine allograft materials compared with the native ACL. Reconstructions were performed using: (1) bone-patellar tendon-bone allograft (BPTB), (2) reconstructive porcine tissue matrix (RTM), or (3) an RTM-polymer hybrid construct (Hybrid). Forces and moments were measured over the entire gait cycle and contrasted at heel strike, mid stance, toe off, and peak flexion. The Hybrid construct performed the best, as magnitude and temporal changes in both anterior knee force and internal knee moment were not different from the native ACL knee. Conversely, the RTM knees showed greater loss in anterior knee force during 2000 cycles than the native ACL knee at heel strike and toe off, with an average force loss o...
The Journal of bone and joint surgery. American volume, 1984
Virtually all types of collagenous tissues have been transferred in and around the knee joint for... more Virtually all types of collagenous tissues have been transferred in and around the knee joint for intra-articular and extra-articular ligament reconstructions. However, the mechanical properties (in particular, strength) of such grafts have not been determined in tissues from young adult donors, where age and disuse-related effects have been excluded. To provide this information, we subjected ligament graft tissues to high-strain-rate failure tests to determine their strength and elongation properties. The results were compared with the mechanical properties of anterior cruciate ligaments from a similar young-adult donor population. The study indicated that some graft tissues used in ligament reconstructions are markedly weak and therefore are at risk for elongation and failure at low forces. Grafts utilizing prepatellar retinacular tissues (as in certain anterior-cruciate reconstructions) and others in which a somewhat narrow width of fascia lata or distal iliotibial tract is utili...
Uploads
Papers by David Butler