Perelman School of Medicine at the University of Pennsylvania

McKay Orthopaedic Research Laboratory

Facilities at McKay Orthopaedic Research Laboratory

The McKay Laboratory of Orthopaedic Surgery Research occupies ~15,500 square feet on the third, fourth, and fifth floors of Stemmler Hall at the University of Pennsylvania and is a shared research facility for the faculty of the Department of Orthopaedic Surgery.The functional units and capabilities are:

Bioengineering

This is an electrically shielded laboratory with a triple-walled, inertial-slabbed equipment room. It is fully equipped for routine and sophisticated materials testing. The primary instrumentation consists of an Instron 8874 Biaxial Servo-hydraulic Testing Machine, an Instron 5543 Electromechanical Testing Machine, two Instron 5542 Electromechanical Testing Machines, two Instron 5848 Electromechanical MicroTester systems, and an Instron ElecroPuls 3000 All-Electric Dynamic Testing Machine. These machines are custom fit with software for test control and data acquisition. A large collection of interchangeable hardware for compression, tension and bending, and maintenance of controlled environment is also available for use with these machines. Additional hardware for the Instron machines can be custom-made in the in-house machine shop, which includes two band saws, a drill press, an end mill, a lathe, and a wide variety of hand tools. All Instron machines are paired with a separate computer that is equipped with custom-written software for the purpose of image capture during testing. A large array of video and camera equipment is available for capturing images during mechanical testing, including: color and black/white CCD video camera backs (both NTSC and PAL formats), and a large variety of lenses for high magnification and wide-angle views, as well as several lens filters. For high-resolution imaging, Basler Scout and Basler Ace cameras provide pixel arrays of 1626 x 1236 and 2048 x 2048 respectively. These black and white cameras have outstanding resolution, providing a dynamic range of 1024 levels of gray. In addition, a system for capturing and analyzing digital images from various microscopes, video systems, and cameras is available. This system includes a positioning device for precise placement of imaging equipment and/or specimens, PC imaging station with a frame grabber capable of 30 frames/second high resolution color imaging, and multiple software tools for image analysis and display. A thickness measurement device is available, composed of a near frictionless LVDT laser probe and platform apparatus capable of directly measuring sample thickness down to 0.01 mm. Two dissecting microscopes are also available for dissection/surgical work, ranging from 4x - 25.2x magnification. Illumination is achieved via heatless fiber-optic light sources. A Leica SM2400 Sledge Microtome, equipped with a Physitemp freezing stage allows for preparation of soft tissue mechanical test samples of uniform thickness. Lastly, a fluoroscope system is present with a five degree-of-freedom tube placement system and a generator for imaging bone and other tissues. The generator is capable of 40-125kV@25-500mA of high-frequency power. The tube has a focal spot of 0.3-1.2 mm for high-resolution imaging of small specimen areas. This system also includes a film processor for on-site development of hard copy images.

Micro Computed Tomography (CT) Imaging

This facility is equipped with 2 state-of-the-art high-resolution micro computed tomography imaging systems. Scanco vivaCT40 is a microCT scanner for in vivo small animal and in vitro specimen scans with nominal resolution up to 10.5 µm. A mobile non-rebreathing anesthesia machine is dedicated for in vivo rodent scans. For ultra-high resolution specimen scans, a Scanco microCT35 is available with a nominal resolution up to 3.5 µm. Each scanner is controlled by a dedicated HP Professional Workstation with an Integrity Server rx2660 (Dual core-2x1.4GHz CPU), 4x8G Ram, 3x146GB and 5x1000 GB Disks, a 24" TFT Monitor, and a 1.6T LTO-4 tape drive. Each system is installed with OpenVMS operation system, microCT measurement program, fast imaging reconstruction, database program, image analyses software, and bone morphometry software.

Computation

The McKay Laboratory contains >80 PC based and Macintosh personal computers. Almost all of these computers are networked via Penn-net for access to the Internet. These computers have the capability for word processing, spreadsheets, statistical analyses, charts and presentation materials, and image analysis. In addition, there are several high performance computers which contain software for programming, data analysis, and finite element analysis (including MATLAB and ABAQUS). Another set of high performance computers are used to control large equipment, acquire data, and acquire and analyze optical images. In addition, several printers (some networked), film recorders, slide scanners, and flatbed scanners are also available in the labs.

Machine Shop

This is a small lab used for the construction and repair of experimental hardware and jigs. The area containing the metal-cutting machinery is vibration isolated from the rest of the floor. A drill press, band saw, grinder, South Bend Lathe, and Bridgeport vertical milling machine with digital readout are located here, as is a reasonable selection of hand and power tools and related accessories. A separate room contains specialized equipment for electrical work, including oscilloscopes, voltmeters, and soldering equipment. A separate closet provides storage space of raw materials.

Histology

This laboratory is located on the 5th floor of Stemmler Hall, and is a shared resource for all McKay lab members. The histology laboratory is equipped with a Reichert-Jung PolyCut S sliding microtome for very large specimens, 2 Olympus CUT 4060 rotary microtomes, a Sorvall MT2-B ultramicrotome, a Tissue-Tek II Tissue Embedding Center, 2 Fisher Flotation Baths, 2 Fisher slide drying boxes and stain sets for H&E, immunohistochemistry & special stains. Specimens of decalcified (or undecalcified) bone specimens and soft tissues may be embedded in paraffin wax (or plastic) or frozen for cryosectioning. General lab equipment includes a balance, heating stir plates, vacuum oven, hybridization oven, stand-alone and canopy fume hoods. Imaging resources include a dual-head Leica BMLB Research Microscope for reviewing slides, a Meiji-Techno dissection microscope, a Zeiss Axioskop 40 FL fluorescence microscope, and a Nikon 50i microscope system with DS-FI1 color digital camera system. Lastly, a Shandon Citadel 2000 automatic tissue processor and a Leica cryostat are available for use.

Microscopy

In an adjacent room on the 5th floor, two additional microscopes are available for specialized imaging. First, for advanced microstructural imaging, a Nikon Eclipse 90i microscope with motorized x-y stages, digital imaging head with epi fluorescence and advances optics for DIC, phase and bright field imaging, and quantitative Bioquant software for bone histomorphometry is available. For additional imaging needs, including live cell cultures, an inverted Nikon TE2000U fluorescent microscope system outfitted with phase and DIC optics, as well as a 100W mercury lamp and standard fluorescent filter sets is available. This system includes a motorized shutter, a motorized z-axis, and an EZ 7Megapixel digital camera all controlled by the NIS-Elements Advanced Research software package, including modules for 4D imaging, real time deconvolution, and time lapse image acquisition. The system is completed with a dual processor workstation with two flat screen panels for image acquisition, storage, and analysis. A computer is connected with a scanner, and Nikon digital camera for capturing, viewing and manipulating of digital images.

A Leica DMLP polarized light microscope with digital image capture/manipulation system and a Lipshaw sliding microtome are also available, as are facilities for autoradiography and photomicrography.

Molecular Biology / Biochemistry / Biophysics

The molecular biology resources are distributed throughout each floor, and include bench space with individual work areas and common areas These areas are well-equipped with fume hoods, analytical balances, top loading balances, pH meters, a conductivity meter, two water purification systems (Millipore), Eppendorf thermomixers, a variety of regular and refrigerated microfuges, low speed and high speed centrifuges (International CU-5000;Sorvall RT-6000B; Beckman J2-HS with JA-10, JA-20, JS-13,JS-75 rotors), several chromatography-style refrigerators, three ultra-low temperature freezers (Forma), microwave, environmental shaking and standard incubators for bacterial cell growth, and multiple water baths. The laboratory also contains specialized work areas for radioactive isotope handling that includes water baths, hybridization incubators (Bellco), microfuge, and a fume hood. Equipment for protein and DNA purification includes sonicators, high-speed tissue homogenizers, a high capacity Virtis lyophilizer, a Labconco FreeZone 2.5 freeze drying system with vacuum pump, a Savant Speed-Vac concentrator, column chromatography, a fraction collecting system, a uv crosslinker, an Invitrogen iPrep DNA isolation system, Syngene gel documentation system, and Gene Pulser I electroporator (BioRad.) Equipment for agarose and polyacrylamide gel electrophoresis includes gel apparatus for horizontal (Hoefer, Pharmacia, and Owl) and vertical (Hoefer and BioRad) gels, electrophoretic transfer (BioRad, Millipore and Invitrogen), and power supplies (Fisher, Stratagene, Hoefer). Additional equipment includes two Nanodrop ND-1000 spectrophotometers, six thermal cyclers (MJR PTC-200, ABI Veriti, Eppendorf MastercyclerPro-S), a Hoefer DyNAQuant 200 fluorometer, an Hitachi Model U-2000 double-beam UV-VIS scanning spectrophotometer with sample sipper, a UV/VIS spectrophotometer (Pharmacia; BioRad), a BioRad 550 microplate reader (415, 490, and 525nm filters), a Biotek Synergy HT multi-detection microplate reader (monochrometer absorbance, luminescence, fluorescence), and three real time PCR systems (ABI 7500, Applied Biosystems StepOnePlus, and Eppendorf Mastercycler ep Realplex). Additional departmental laboratory rooms contain a dark room with Konica film processor SRX-101A for X-ray film developing, a UV light box (VWR), and a fluorometer (Hoefer), a LI-COR Odyssey Infrared Imaging System pro for Western Blotting detection, plate detection, and whole animal imaging, and a Biotek Synergy 2 microplate luminometer with 2 injection ports. A Beckman Coulter Biomek 2000 Laboratory Automation Workstation (with associated controllers) is available for high throughput liquid dispensing and mixing in 96 and 384 well plates.

Specimen Preparation

There are two operating room-style facilities; each equipped to accommodate multiple procedures simultaneously. The first room has adjustable tracked operating room lights with variable light intensities, and two hydraulic stainless steel operating tables. The second room has a 3-foot Envirco laminar flow tabletop clean bench for the sterile dissection and cell and tissue preparation. Both room contain a wide range of surgical instruments and specialty power tools such as arthroscopy equipment, air drills, surgitomes, and oscillators which are fully sterilizable for use in animal surgery and 'clean' dissections. Sterilizing equipment for preparation of instrument packs and surgical clothing are on hand. In addition, an array of refrigerators and freezers are available for tissue storage.

Tissue Culture

The tissue culture laboratory is located on the 5th floor of Stemmler Hall and consists of six rooms, as well as a walk-in cold room, and a walk-in -10oC Freezer. The distribution of rooms include a general hall with -20 freezers and refrigerators, four Thermolyne Cryotanks (models Locator 8, 6, 4, and Jr.), two Cryosafe Protector Plus Cryotanks (models APP1 and APP2), and Liquid Nitrogen source tanks. There are two rooms for tissue culture. The smaller contains two four foot biosafety cabinets: one Forma class A/B3 Model 1184 and one Forma Class A2 Model 1284. Additional equipment includes a counting scope, an Eppendorf model #5702 centrifuge, two Sanyo MCO-17AC, two Forma model 3130, and two Forma model 3110 water jacketed CO2 incubators. The second larger room contains three biosafety cabinets (2 four foot, 1 six foot): one Forma class A/B3 Model 1184, one Forma Class A2 Model 1284, one Thermo Electron Class A2 model 1376 and 12 incubators: ten Forma model 3110 and two model 3130 water jacketed CO2 incubators. Two liquid CO2 tanks and a Victor 1000 Liquefied Gas Automatic Switchover Manifold centrally supplies CO2 to all incubators. Two staging areas contain a large-capacity refrigerated Eppendorf centrifuge mode l#5810, a Nexcelom T4 automated cell counter, two Nikon TS100 inverted microscopes. Live cell imaging can be carried out on a one of the Nikon scopes (with mercury lamp and GFP filter set) equipped with a Nikon DS-Fi1 color digital camera and controller. Adjacent rooms include, one 'clean' dissection room and one microscopy room, as noted above. There are water baths, mini centrifuges, a microwave, vortexes, and storage areas throughout the area for general use.