by, tanner jones, andrew gloe, michael grabarits, hoi wai...
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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: NucleiRed CytoplasmPale Red ErythrocytesOrange Collagen FibersDeep
Blue
More Specifically : Keratin Orange CartilageBlue Bone MatrixDeep Blu Muscle FibersRed
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 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 of 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
open
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 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 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
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