Facilities at McKay Orthopaedic Research Laboratory
The McKay Laboratory of Orthopaedic Surgery Research occupies >22,000 square feet on the third floor of Stemmler Hall at the University of Pennsylvania.
This space was fully renovated (occupancy 10/2018) as part of a $120M building-wide renovation of Stemmler Hall that resulted in an expanded, efficient, and modern research space.
The McKay Laboratory is a multi-disciplinary, shared, research facility that undertakes a broad range of musculoskeletal research investigations. In addition to facilities for molecular and cell biology research, laboratories additionally include shared facilities for imaging and histology, biochemical analysis of connective tissues, and bioengineering-based assessment of tissue structure and function.
The functional units and capabilities are described in detail below.
Mechanical Testing
Mechanical testing equipment at McKay is housed in several adjacent rooms that are 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, an Instron ElecroPuls E3000 all-electric dynamic fatigue testing machine, a Bose Electroforce 5500 microtester, and an ADMET eXpert 4901 custom uniaxial testing machine. There also exists a Bose 3550 Axial Torsional Tester and a Bose 3330 Series II Axial Tester (at the TMRC, an 8-minute walk from McKay), as well as a Tekscan pressure sensor system for the measurement of pressures between articulating joints. A Bruker Bioscope Catalyst Atomic Force microscope is located in an adjacent building (and managed by McKay) for micro- and nano-indentation testing of soft and hard tissue specimens. Additional instrumentation at McKay includes two custom compression testing devices for confined and unconfined testing, a microtorsion instrument that applies combined axial compression and torque, an adaptation unit and fixturing for shear testing using the Instron 5543 and 5542 systems, a micro-indentation rig with universal joint for specimen positioning, a custom cross-polarizing system for assessing tissue collagen fiber organization during loading, and an Instron actuator and load cells added to the 8874 for a third axis of loading in bending orientations. Other custom equipment includes compression and tension devices that can be mounted on inverted microscopes for imaging cellular and sub-cellular deformation and quantifying local mechanics.
Each of the testing machines is custom fit with software for test control and data acquisition. For the larger testing systems, a collection of interchangeable hardware for compression, tension and bending, and maintenance of controlled environment are available. Additional hardware can be custom-made in the in-house machine shop (described below). Each system includes a positioning device for precise placement of imaging equipment and/or specimens, and a PC equipped with custom-built software for real-time magnified image display and post-processing analysis. An array of video and camera equipment is available including color and monochromatic digital cameras capable of capturing high resolution images at a frame rate of 15 Hz to 150Hz, a variety of lenses for high magnification and wide-angle views, as well as several lens filters. These cameras can be integrated with several different types of available monitors and computers to record changes in geometry throughout mechanical testing as well as monitoring position of markers for subsequent digital image correlation of local deformation. Additionally, a motion tracking system can be used for three-dimensional tracking of segments throughout the test volume during mechanical testing. Six dissecting microscopes (Leica DM205c, S9i, M205C) are also available for dissection/surgical work, ranging from 4x - 25.2x magnification. Illumination is achieved via heatless fiber-optic light sources coupled with LED ring light systems. A Leica SM2400 Sledge Microtome, equipped with a Physitemp freezing stage allows for preparation of soft tissue mechanical test samples of uniform thickness. Two laser-based thickness and area measurement devices that utilize a CCD laser positioned over an x-y translation stage generates surface plots to measure sample thickness with an accuracy of 0.16 microns. An Orthoscan FD Mini C-arm fluoroscope that can be rotated 360° in three planes while recording digital still images or video is also available. The fluoroscopy system can be used to measure geometries of bone and joint spaces (i.e., disc height) prior to mechanical testing.
Micro Computed Tomography (CT) Imaging
There are three µCT systems within the McKay labs. The Scanco vivaCT 80 (Scanco Medical AG, Bassersdorf, Switzerland), acquired in 2018, are preclinical systems designed for in vivo scans of small rodents (mice and rats). These scanners provide an image voxel size down to ~10 μm. The vivaCT 80 can accommodate scans of larger regions of interest such as ex vivo scanning of large samples and in vivo scan of abdominal region of rats (e.g., lumbar spine, abdominal fat and muscle, and other internal organs). The Scanco µCT 35 and µCT 45, acquired in 2012 and 2019, respectively, are designed for small specimens with the best voxel size at 2.5 μm. The µCT 45 is equipped with a 20-sample holder carousel that automates batch scans and supports rapid phenotyping of large quantities of samples. Each of the scanners are controlled by a dedicated HP Professional Workstation Integrity server. All servers use the same version of software for image acquisition, analysis, visualization, backup, and storage. Additionally, a fifth HP Integrity workstation and a GPU reconstruction workstation are incorporated to speed up image processing and data analysis, and four associated external data towers provide a total storage capacity of 145 TB. This setup facilitates the management of the scanners, user training, data backup, and data exchange among different scanners. In addition to the µCT units, this facility is also equipped with two rodent anesthesia machines (Supera Anesthesia, Clackamas, OR) for in vivo imaging studies and a perfusion system which includes a perfusion pump (Econo Pump, BioRad, Hercules, CA) and a syringe pump (Advance Series 1200 Infusion System, Cell Point Scientific, Gaithersburg, MD), for contrast-enhanced vascular cast imaging. Additionally, the vivaCT80 is equipped with a mechanical testing stage (Scanco Medical AG) that facilitates real time imaging of microstructure changes in specimen under load. This facility also contains five HP Professional Workstation Integrity Servers rx2660 that operate µCT scanners and perform image reconstruction and analysis. In addition, there are >15 high performance computers that contain software for programming, images registration, image analysis, and finite element analysis (including MATLAB, ITK, Visual C++, SolidWorks, Hypermesh, FE Bio, and ABAQUS).
Computation
The McKay Laboratory contains >100 PC based and Macintosh personal computers. Almost all of these computers are networked via Penn secured internal network 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 3 high-performance computers that contain software for programming, data analysis, and finite element analysis (including MATLAB, ABAQUS, SolidWorks, GraphPad, Systat, Adobe Creative Cloud, Microsoft Office 365, Makerbot Desktop, Vic 2-D). These are available to all lab members via secure remote access. Another set of high-performance computers are used to control large equipment, acquire data, and acquire and analyze optical images. In addition, several networked printers, and flatbed scanners are also available throughout the labs.
Machine Shop
This is a dedicated lab room that is used for the construction and repair of experimental hardware and jigs. A drill press, band saws (one horizontal and one vertical), grinder, Grizzly Lathe, belt sander, and Bridgeport vertical milling machine with digital readout are located here, as is a reasonable selection of hand and power tools and related accessories. The room also contains specialized equipment for electrical work, including oscilloscopes, voltmeters, and soldering equipment. A separate closet provides storage space for raw materials.
A separate room contains two fused-deposition modeling (FDM) 3-D printers (FlashForge Creator Pro) that provide for rapid creation of plastic prototypes and radio-transparent jigs. A FormLabs Form3 stereolithographic 3D printer has been recently acquired that can be used to fabricate complex parts out of autoclavable materials. This room also houses a MarkForge Mark II FDM printer capable of producing high-strength parts by combining carbon-fiber infusion with a proprietary high-strength thermoplastic called Onyx, one Ultimaker S3 Fused Filament Fabrication 3D Printer, and two Prusa i3 MK3 3D printers.
Histology
The McKay Laboratory contains a centralized facility containing sophisticated resources and equipment to support histological processing and analysis of the complete range of musculoskeletal tissues, including intervertebral disc, bone, cartilage, fibrocartilage, tendon and muscle from large and small animal models, human tissue samples, and tissue-engineered constructs. Conventional techniques supported by this facility include paraffin, frozen and plastic processing, embedding, sectioning and histochemical staining, as well as imaging and analysis, and more advanced and sophisticated techniques such as immuno-histochemistry and histomorphometry. Cutting edge techniques including cryofilm for cutting undecalcified sections and RNAscope in situ hybridization assays optimized for musculoskeletal tissue sections are also available. Specific equipment for specialized functions is detailed in the following sections.
Paraffin Histology: Key equipment includes four paraffin microtomes (a ThermoFisher Shandon Finesse 325, an Olympus CUT 4060, a Leica RM2125, and a Reichet-Jung 2030), a Shandon/Lipshaw paraffin dispenser, a Tissue-Tek II tissue embedding center, three ThermoFisher flotation baths, five slide drying devices, and a ThermoFisher Excelsior AS automated tissue processor. Frozen Histology: Key equipment includes three cryostat microtomes (a Leica CM1950 and two ThermoFisher NX70s), an Instamedic CryoJane ECU tape system (for undecalcified mineralized tissues). Additionally, all three cryostats support the cryofilm method for cutting undecalcified mineralized tissues. Plastics Histology: Key equipment includes a Reichert-Jung PolyCut S sliding microtome, a Craftsman belt/disc sander, a Buehler MetaServ 20 Grinder-Polisher, and a dedicated fume hood for processing samples into methyl methacrylate. Imaging and Analysis: Key equipment includes a dual-headed Leica BMLB teaching microscope, a Meiji-Techno dissection microscope, a Nikon 50i microscope system with DS-FI1 color digital camera system, and a Zeiss Axioskop 40 FL fluorescence microscope. Quantitative Bioquant Osteo Analysis software for bone histomorphometry is available on two dedicated workstations, and an additional workstation with Osteometric Osteomeasure Software is available. Two Nikon student “wet” microscopes are used for monitoring section preparation. For automated slide scanning, two Axio Scan.Z1 imaging systems are available. These systems are highly automated digital bright field and epifluorescent slide scanners capable of imaging up to 100 slides at a time at up to 20X resolution. These systems are equipped with solid state LED light sources and 10 channel fluorescent filter turret capable of imaging the full spectrum available for epifluorescence. Staining and General Lab Equipment: Key resources include a dedicated fume hood fully equipped for routine and special histochemical staining and immunohistochemistry, a Bellco Autoblot microhybridization oven, and general lab equipment including a balance, heating stir plates, a 37ºC oven, a vacuum oven.
Microscopy
Several microscopes are available throughout the laboratory for specialized imaging. First, for advanced microstructural imaging, two Nikon Eclipse 90i microscope with motorized x-y stages, digital imaging head with epi-fluorescence and advanced optics for DIC, phase, and bright field imaging are available. These microscope are controlled by the NIS-Elements Advanced Research software package, including modules for 4D imaging, real time deconvolution, and time lapse image acquisition. 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 a high resolution pco.edge 4.2 LT scientific CMOS 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. Also available is a Leica DMLP polarized light microscope with digital image capture/manipulation system. Additional microscopes available for specialized imaging include a Zeiss Axio imager D2, Zeiss Axio Zoom.V16 microscope, Dual Lit Halogen Trinocular Stereo Zoom Microscope with Camera, and a Zeiss AxioOberver microscope with live cell imaging platform. Two Leica Ultramicrotomes are also available to prepare specimens for TEM analysis.
Molecular Biology / Biochemistry / Biophysics
The molecular biology resources are distributed throughout the department and include bench space with individual work areas and common areas. These spaces are well-equipped with analytical balances, top loading balances, pH meters, a conductivity meter, Eppendorf Thermomixers, a variety of regular and refrigerated microfuges, a Thermo Legend XTR low to high speed centrifuge with F115-8X50cy and TX-1000 rotors, a Sorvall X4R Pro-MD, 120V TX-1000 centrifuge, several chromatography-style refrigerators, >40 -20°C freezers and refrigerators, 10 ultra-low temperature freezers (Forma and Thermo), microwaves, environmental shaking and general incubators (two Thermo Heratherm), hybridization ovens (Boekel Big Shot III) and multiple water baths. Equipment for protein and DNA purification includes sonicators, high-speed tissue homogenizers, a Labconco FreeZone 2.5 freeze drying system with vacuum pump, column chromatography, a fraction collecting system, a UV crosslinker, a BioChain Anaprep 12 DNA isolation system, and a 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 three Nanodrop spectrophotometers (ND-1000, ND One-C, ND-2000) and ten thermal cyclers (Applied Biosystems Veriti, Eppendorf MastercyclerPro-S, Biorad T100, Biorad C1000, Biorad S1000, Applied Biosystems Simpliamp, Eppendorf Mastercycler Nexus Gradient, Applied Biosystems ProFlex PCR System). Other equipment includes a Hoefer DyNAQuant 200 fluorometer, a 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, fluorescence, luminescence), three Real-Time PCR systems (QuantStudio6 Flex, QuantStudio6 Pro, and Eppendorf Mastercycler ep Realplex). There is also a Li-Cor C-Digit Chemilluminescent Imaging System, and two ChemiDoc XRS+ gel documentation systems with Image Lab Software. Additional departmental laboratory rooms contain a dark room with 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 two iBlot transfer systems. Other specialized equipment available include a Grant Instruments 5L Boiling Water bath, ECM 830 Generator with 2 needle array, Precellys evolution homogenizer, Qiagen TissueLyser LT, and SPEX SamplePrep Cryogenic Grinder.
Specimen Preparation
There are two operating room-style facilities; each equipped to accommodate multiple procedures simultaneously. Each room has adjustable operating room lights with variable light intensities, and a hydraulic stainless steel downdraft operating table. One room has a 3-foot Envirco laminar flow tabletop clean bench for the sterile dissection and cell and tissue preparation. Both rooms 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. An Isomet low speed saw enables the preparation of soft and hard tissue mechanical test samples of uniform thickness. Sterilizing equipment for preparation of instrument packs and surgical clothing are on hand.
Tissue Culture
The tissue culture laboratory consists of 7 rooms. The distribution of rooms includes a general hall with -20oC freezers and refrigerators, nine Thermolyne Cryotanks (models Locator 8, 6, 4) and three Liquid Nitrogen source tanks. There are four workrooms for tissue culture. Throughout these rooms are 6 four-foot Thermo Class A2 model 1375 Biosafety Cabinets, 1 six-foot Thermo HeraSafe KS18 Biosafety Cabinet, and 1 HypOxystation H35 Hypoxygen Workstation. There are a total of 21 CO2 Incubators within the tissue culture rooms: 12 Forma model 3110, two Forma model 3130 CO2/O2, and 2 Forma model 4110 water jacketed incubators, 3 Heracell 150i, and 2 Forma Steri-Cycle model 370 incubators. An Airgas 240 Liquid Cylinder Automatic Switchover Manifold with connections for up to (6) 160 Liter Cylinder (350 PSI) Industrial Grade CO2 tanks centrally supplies CO2 to all incubators. A centralized staging area adjacent to the 4 work rooms contains 2 large-capacity refrigerated Eppendorf centrifuge models 5810/5810R. The facility also houses a Nexcelom T4 automated cell counter, two Nikon TS100 inverted microscopes, and a Nikon Ts2 microscope. Live cell imaging can be conducted on one of the Nikon scopes (with mercury lamp and GFP filter set) equipped with a Nikon DS-Fi1 color digital camera and controller. Two adjacent rooms include one ‘clean’ dissection room and one microscopy room which contains the Nikon TE2000U fluorescent microscope described in the Microscopy section. There are also water baths, mini centrifuges, a microwave, vortexes, and storage areas throughout the facility for general use.
Additional Equipment and Facilities
Other resources available in the McKay laboratory include a Steris Reliance 400XLS Laboratory Glassware Washer, two steam sterilizers (AMSCO Lab250 Small, AMSCO Century Medium), five Millipore Integral 10 water purification systems, 15 chemical fume hoods, three cold rooms, and two Flow Sciences Ventilated Safety Enclosures. A Material Fabrication room with 4 movable Fumex Extractor units and controlled temperature/humidity houses four custom electrospinning devices with power supplies. Additional equipment includes a custom capillary capsule fabrication station (with scope, high speed camera, and syringe pumps), and a Narishige International Microforge MF2 (with 50 & 100X lens set). There is also a dedicated surgical procedure room approved for non-USDA rodent procedures, including survival surgeries, with a movable operating table and Carl Zeiss Opmi CS / NC 31 Contraves Floor stand Surgical Microscope System. Other dedicated spaces for small animal work contain a custom Instrumented walkway for rodent gait and ground reaction force measurement and custom treadmills for rodent activity and overuse studies.
Updated Jan 2022