By, Tanner Jones, Andrew Gloe, Michael Grabarits, Hoi Wai Chau, and - PowerPoint PPT Presentation

By, Tanner Jones, Andrew Gloe, Michael Grabarits, Hoi Wai Chau, and Sarah Bradner

 University of Gothenburg, The Sahlgrenska Academy, Institute of Biomedicine,  Hakan Nygren  Cecilia Eriksson  Katrin Richter  Karin Ohlson  Elos Medical AB, Backendalsvagen  Nicklas Billerdahl  Mattias Johansson

 PhD. Histology; Histologiska Institutionen at Gothenburg University  Thesis: Immunoenzyme methods;.  Head of the Imaging Mass Spectrometry Research Group at University of Gothenburg as of 2008  Focus on Histological Analysis by use of TOF-SIMS  Ph.D Advisor of Cecelia Eriksson and Katrin Richtor

 Doctoral Degree: Medicine/Histology  Thesis: Interactions between whole blood and TiO2 surfaces with focus on adhesion and activation of polymorphonuclear granulocytes  Post-Doctoral Work(2003): The University of Gothenburg  Most Current(2011) : Head Life Sciences Deptartment, Biomedicine, University of Skövde  16 peer reviewed publications

 Masters of Science in Biology at the University of Rostock, Germany  Doctorate: University of Gothenburg  5 Peer Reviewed Publications

 Not one of Professor Nygren’s doctoral students.  Has no citations on either PubMed or Wiley Online Library  Could be a lab tech or a just the result of translational butchery.

 Elos Medtech- self described as “one of Europe’s leading development and production partners for medical technology products and components.”  Based in Timersdala Sweden  Appears their involvement in the project was concerned with the design and supply of the experimental materials.  No conflict of Interest statement

 TOF-SIMS (Time of Flight Secondary Ion Mass Spectrometry) 
 : A method of imaging, which allows for the characterization of a specimen’s chemical:  composition  distribution  depth profile.  ToF-SIMS is particularly useful in that does not depend on probes or antibodies which would impose their own unique physical and chemical limitations on what can be imaged. TOF-SIMS imaging limited only by what can be ionized in a single sample analysis session.  The great challenge lies in sample preparation  Imaging must performed under ultra high vacuum conditions.  Samples most be freeze dried or freeze fractured to keep them as close to native conditions as possible  http://www.youtube.com/watch?v=8wzZcsNk_80

 Cortical bone: High density, mature osseous tissue. Cortical bone facilitates support of the whole body and protection of the organs while also providing levers for movement  Passivation: The process of intentionally producing a layer of corrosion on the surface of a biomaterial for the purpose of reducing its surface reactivity.  Bone Resorption : The process by which osteoclasts break down bone into its constituent minerals.  
 Anodic oxidation: 
 An electrolytic passivation method in which the treated material forms the negative terminal of an electric circuit.  Mallory’s Trichrome Stain: Commonly Used for the identification of connective tissue

 Cell Members affected: More Specifically :  Nuclei  Red Keratin  Orange  Cytoplasm  Pale Red Cartilage  Blue  Erythrocytes  Orange Bone Matrix  Deep Blu  Collagen Fibers  Deep Muscle Fibers  Red Blue

 Post fracture  Bleeding, blood coagulation, hematoma  Inflammation  Soft Callus Formation  Hard Callus Formation  Bone Remodeling

 Occurs immediately after injury  Extravascular blood cells form a blood clot  All the cells within the blood clot degenerate and die  Thrombin and Growth Factors are released by activated leukocytes  Activate fibroblasts aggregate and form granulation tissue  Platelets in the hematoma serve as chemotaxins for osteogenic cells  Filled with vascular endothelial growth factor (VEGF)  Involved in angiogenesis and bone t

 Stabilizes the fractured area with granulation tissue and fibrocartilage  Spongy material  Callus will keep expanding until fracture is stabilized  Internal and External callus  Once stabilized blood vessels will invade the callus

 Very narrow compact region found in the fracture union  Internal callus has high cellular density  Very compact region  Found adjacent to the fibrin clot (hematoma)  Contains cells of endosteal origin  Large quantities of Fibrin and cartilage

 External callus is larger, but low cellular density  External callus is adjacent to bone marrow  Cells are derived from progenitor cells found in the periosteum  Polymorphic MSC and osteoblasts are responsible for early synthesized bone matrix  Primarily made of woven bone and cartilage

 Vascular density in the callus increases  Endochondral ossification of spongy bone into woven bone  Vesicles are released by osteoblasts  Initiates tissue mineralization  Release hydroxyapatite crystals  Organic components of bone are mineralized  Type I collagen fibrils and noncollagenous matrix proteins  Convert less stable spongy bone into stronger woven bone

 Over laps with the hard callus formation  Hard callus is still bulky and needs to be remodeled into previous uninjured state  Woven bone is replaced over time with compact lamellar bone  Bone becomes more organized in parallel fibers  VEGF are the growth factors that regulate remodeling  Attracting endothelial cells and osteoclasts  Stimulates osteoblast differentiation  Osteoclasts remove woven bone, and osteoblasts lay down lamellar bone

 Bone healing is a process that does not result in scaring  Insertion of implants leads to complete healing  Poorly inserted implants can lead to instability and eventually failure  Instability causes fibrous encapsulation instead of implant bone contact  Implants that extend into the marrow cavity cause bone tissue to remain in the marrow cavity  This is not observed in normal fracture healing

 Why does the presence of a titanium plate placed in the fractured union lead to the formation of bone tissue in the marrow cavity?

Thickness: 1mm Diameter: 2.5mm Threaded hole with 0.8mm diameter Grade 1 Unalloyed titanium, low oxygen. Grade 2 Unalloyed titanium, standard oxygen. Grade 2H Unalloyed titanium (Grade 2 with 58 ksi minimum UTS). Grade 3 Unalloyed titanium, medium oxygen. .. . . Grade 38

 Passivated discs in 4.9M HNO 3 for 20 min  Washed in alcohol Anodic Oxidation to grow porous oxides Platnium band (cathode) titanium+discs (anode) HF (hydrofluoric acid) + H 2 SO 4 (Sulfuric acid)= strong oxidizing agent  B11 HF (hydrofluoric acid) + H 2 SO 4 (Sulfuric acid) + H 3 PO 4 (phosphoric acid)  G4 and G1 Rinsed in deionized water  alcohol based washing

 Time-of-flight secondary ion mass spectrometry -positive and negative spectra recorded  Auger Electron Spectroscopy (AES): provides elemental analysis of surfaces by measuring energies of backscattered electrons. -very sensitive -can monitor surface cleanliness -compositional analysis of specimens in surface region http://www.cem.msu.edu/~cem924sg/Topic10.pdf

 Surfaces were photographed  SEM images segmented  Measured mean pore diameter, #pores/µm 2 , and surface porosity

Shaving and Male cleaning of Anesthesia Sprague Muscle and calves with with Isofluran Dawley rats bone iodine Baxter (350-500g) exposed by 2cm-long lateral 1mm Muscularis Incision, incision diameter tibialis anterior rinse and Hole drilled aside and Implant in facies periosteum placed in lateralis of open each tibia tibia Buprenorphin Skin sutured Post-op below dermis Free post-op and movements epidermis

The surgical procedure used to insert the implant consisted of drilling a 1mm diameter hole in the facies lateralis of the tibia with a low speed drill. How could drilling method detrimentally impact the rate of implant healing and osseointegration?

 Incisions made in bone  Left to heal and no implant

 Animals sacrificed at 4,7 and 14 days  Bone site of implantation was extracted  Samples fixed in PBS for 3 days  Decalcified for 2 weeks in 0.5% paraformaldehyde in PBS (makes bone flexible and easier to analyze)  Samples were rinsed in water for 15 min  Samples dehydrated in graded series ethanol  Imbedded in Histowax imbedding medium  Cut and mounted on Superfrost plus glass slides  Stained with Mallory’s trichrome

 Stain tissue photographed with microscope  Area measured  Percent of bone contact with implant relative to blood and connective tissue measured  Thickness of bone in contact was not measured

 ANOVA post hoc test: examining of data after the experiment to look for patterns. Statistical test performed once pattern is found.  Significance set to p<0.05

 B11 was processed using H 2 PO 4  Contains low P component compounds  G4 and G1 were processed using H 2 PO 4 andH 2 SO 4  Phosphorus is the second abundant mineral in the bone.  Used for development and maintenance of healthy bones

How can the different in surface compound affects the implant healing results?