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

• f 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

• f 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

• r 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:

 NucleiRed  CytoplasmPale Red  ErythrocytesOrange  Collagen FibersDeep Blue

More Specifically : Keratin  Orange CartilageBlue Bone MatrixDeep Blu Muscle FibersRed

 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

• xygen.

Grade 2H Unalloyed titanium (Grade 2 with 58 ksi minimum UTS). Grade 3 Unalloyed titanium, medium

• xygen.

.. . . Grade 38

 Passivated discs in 4.9M HNO3 for 20 min  Washed in alcohol

Anodic Oxidation to grow porous oxides Platnium band (cathode) titanium+discs (anode) HF (hydrofluoric acid) + H2SO4 (Sulfuric acid)= strong oxidizing agent B11 HF (hydrofluoric acid) + H2SO4 (Sulfuric acid) + H3PO4 (phosphoric acid) G4 and G1 Rinsed in deionized water alcohol based washing

• Auger Electron Spectroscopy

(AES): provides elemental analysis of surfaces by measuring energies

• f backscattered electrons.
• very sensitive
• can monitor surface cleanliness
• compositional analysis of

specimens in surface region

• Time-of-flight secondary ion mass spectrometry
• positive and negative spectra recorded

http://www.cem.msu.edu/~cem924sg/Topic10.pdf

 Surfaces were photographed  SEM images segmented  Measured mean pore diameter, #pores/µm2, and surface porosity

Male Sprague Dawley rats (350-500g) Anesthesia with Isofluran Baxter Shaving and cleaning of calves with iodine Muscle and bone exposed by 2cm-long lateral incision Muscularis tibialis anterior aside and periosteum

• pen

1mm diameter Hole drilled in facies lateralis of tibia Incision, rinse and Implant placed in each tibia Skin sutured Buprenorphin below dermis and epidermis Free post-op movements

Post-op

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

• sseointegration?

• 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

• nce pattern is found.

 Significance set to p<0.05

 B11 was processed using H2PO4

 Contains low P component compounds

 G4 and G1 were processed using H2PO4 andH2SO4  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?

How might the long-term effects vary among the four surface properties control, B11, G4 and G1. If these surfaces were studied long-term, what may be another useful variable to quantify besides bone-to-implant contact?

Figure 4. (a-e) Normal healing after (a) 0 days, (b) 4 days, (c) 4 days (close- up), (d) 7 days, (e) 14 days.

 4 Days

 Formation of soft callus and new bone

 7 days

 Formation of woven bone and hard callus  Bone resorption with in the marrow

 14 days

 Woven bone has been replaced with more mature bone

Figure 4. (f-i) Implant healing of the control surface after (f) 4 days, (g) 7 days, (h) 14 days.

 4 Days

 New bone formation adjacent to the endosteum of the cortical bone

 7 Days

 Woven bone surrounding the implant

 14 Days

 Woven bone on the implant surface has been replaced by lamellar bone

 Within 14 days, bone formation, resorption, and maturation had taken place in both fracture and implant healing

• overtime, bone and marrow will be completely

restored in fracture healing

• Osseointegration is necessary for implant

stability (imbedding in layer of bone good, fibrous tissue formation around implant bad)

• Excessive bone resorption also bad

REFERENCE STUDY THIS STUDY Hanawa et al. in a similar rat Ti implant study found:

• Initial bone formation in the marrow

followed by resorption

• After 18 days, bone stayed in a thin

line around implant

• After 7 days, some bone found in

close contact with implant surface + bone formation in marrow space around implant

• Bone resorption in marrow between

7-14 days

• After 14, bone stayed close to

implant Ushida et al. drilled holes in bones similar to this study but no implants inserted:

• Bone formation in marrow after 5-7

days

• After day 11, bone gradually replaced

with marrow (resorption)

• “small islands of bone” (bone

formation) seen after 4 days in both implant/fracture healing

• Resorption at 14 days

Takeshita et al. - Ti implants in rat tibia studied after 28 and 730 days.

• Found bone thickness increases

after implant but established early in

• After resorption of callus bone,

implant surrounded by thin incomplete layer of bone after 2 weeks same layer seen in

Reference Study THIS STUDY Medard et al. found:

• the amount of bone in rats

decreased where implant exposed to marrow between 7 & 21 days

• mature bone stayed close to surface
• After 14 days, initially formed bone

resorbed

• Mature bone remained close to

surface

• This study in accord with previous research

regarding healing process

• Resorption is very important for strong

implant attachment ** “Reducing Desorption” of bone early on could be an asset for developing implants that integrate better

http://www.intechopen.com/source/html/29733/media/image2.jpg

• Other studies have shown more porous

implants integrate better with bone in the long term (6-12 weeks) in rabbits

• This study showed implant healing was not

significantly affected by implant porosity since all implants had similar bone contact

 Dhert et al. agrees: “biology rather than implant properties” is main factor in early implant healing

 Fractures and implant injuries both heal in similar ways in terms of structure and rate  After 14 days, the implant was enveloped in lamellar (strong) bone and the marrow restored  Porosity of titanium implant did not affect bone integration after only 7 days