Orthopedic Conditions That Require Regeneration Of Bone And Enhancement Of Bone Healing Include Substantial Bone Devitalization And/or Loss From Trauma (E.G., Highly Comminuted Fractures) Or From Resection Of A Neoplasm, Arthrodesis, Spinal Fusion, Nonunion Or Delayed Union Fractures, Arthroplasty, Fragment Gap Defects During Corrective Osteotomy, Reduced Healing Potential From Local Or Systemic Disease (E.G., Hyperadrenocorticism, Hypothyroidism, Neoplasia), And Patients With A Poor Osteogenic Potential (Older, Debilitated, Or Small-breed/Toy-breed Dogs). By DR.Kambiz Yousefi Kambiz.u3fi@me.com

1. Bone Grafts and Substitutes
Karl Herbert Kraus and Steven A. Martinez
D R . K a m b i z Yo u s e f i
P r e s e n t e d B y :

2. Orthopedic Conditions That Require Regeneration Of Bone And Enhancement Of Bone Healing Include Substantial Bone
Devitalization And/or Loss From Trauma (E.G., Highly Comminuted Fractures) Or From Resection Of A Neoplasm,
Arthrodesis, Spinal Fusion, Nonunion Or Delayed Union Fractures, Arthroplasty, Fragment Gap Defects During Corrective
Osteotomy, Reduced Healing Potential From Local Or Systemic Disease (E.G., Hyperadrenocorticism, Hypothyroidism,
Neoplasia), And Patients With A Poor Osteogenic Potential (Older, Debilitated, Or Small-breed/Toy-breed Dogs).
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3. MESENCHYMAL STEM CELLS
The Embryo Forms From A Single Cell That Gives Rise To Cells That Have An Increasingly Narrower Spectrum Of
Differentiation.
The Mesenchymal Stem Cell Is A Stage Along This Path Of Differentiation By Which The Cell Can Follow
Osteoblastic, Chondroblastic, Adipocytic, Tenocytic, Or Myoblastic Lineages.
A Population Of These Stem Cells Is Preserved In The Adult In A Variety Of Tissues; The Most Accessible Sources
Include The Cambium Layer Of The Periosteum, The Bone Marrow, And Fat. Although The Number Of Stem Cells
Diminishes With Age, Stem Cells Are Present Throughout Life And Can Be Retrieved, Isolated, And Culture Expanded.
Mesenchymal Stem Cells Can Be Manipulated In Vitro Or In Vivo To Follow Osteoblastic Lineages.
Cultured mesenchymal stem cells. These cells are
the building blocks of embryologic bone
formation, bone healing, and bone regeneration.
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4. 4
MESENCHYMAL STEM CELLS
The periosteum of sheep tibia. (a) Magnification 250 and (b)
magnification 25. Photomicrograph of normal
periosteum attaching to bone. Periosteum consists of two
clearly divided layers: osteogenic, cambium (K) and fibrous (F)
layer. Periosteal surface (P) adjacent to the cortex.

5. CLINICAL APPLICATION OF BONE GRAFT TECHNIQUES
Bone Grafting Techniques And Applications Are Based On Knowledge Of Bone Formation And Healing. This
Includes Information On Mesenchymal Stem Cells, Growth Factors, And Bone Matrix Development. From
Knowledge Of Bone Formation, Basic Tenets Of Bone Regeneration And Grafting Can Be Developed. These
Are Divided Into Four Classification Strategies: OSTEOGENESIS, OSTEOINDUCTION,
OSTEOCONDUCTION, And OSTEOPROMOTION. The Goal Of All Bone Grafting Techniques Is To Use As
Many Of These Strategies As Possible To Enhance Bone Healing.
• O S T E O G E N E S I S
• O S T E O I N D U C T I O N
• O S T E O C O N D U C T I O N
• O S T E O P R O M O T I O N
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6. O S T E O G E N E S I S
• A Graft That Directly Supplies And Supports Bone-forming Cells Is Termed Osteogenic.
• Fresh, Autogenous (Autogenic Or Autologous) Cancellous Bone Grafts Are The Best Example Of An
Osteogenic Graft And Are The Gold Standard For Bone Regenerative Materials.
• Fresh, Autogenous Cancellous Bone Grafts Revascularize More Rapidly Than Fresh Cortical Autogenous Bone
Grafts, Allowing Survival Of Some Transferred Cells
• The Live Cells Found In Fresh, Autogenous Cancellous Bone Grafts May Begin Producing New Bone Almost
Immediately, Which Is An Advantage Compared To Other Types Of Grafts.
• Osteogenesis Associated With Fresh Autogenous Cancellous Bone Grafts Can Be Initiated As Early As 5 Days
After Implantation, With Maximum Osteogenesis Occurring 8 Weeks After Transplantation
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7. O S T E O G E N E S I S
• Another example of an osteogenic material is bone marrow.
• Bone marrow aspirates contain mesenchymal stem cells—that is, cells already committed to osteogenic or
chondrogenic lineage—and some biologically active proteins that stimulate bone regeneration in a similar manner to a
naturally occurring fracture hematoma.
• Many biologically active proteins are released in situ by activated platelets during their degranulation reactions.
• Although bone marrow contains osteogenic elements, it does not demonstrate the same magnitude of osteogenesis
observed with autogenous cancellous bone grafts.
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8. O S T E O G E N E S I S
Likely Factors For This Finding Include The Following:
1) Bone Marrow Aspiration Results In A Varying Quality Of Bone Marrow, Depending On Both The Aspiration
Technique Used And The Patient;
2) Osteoprogenitor Cell Content May Be Low
3) Bone Marrow Lacks The Necessary Scaffold, Or Osteoconductive Material, To Be Efficacious On Its Own.
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9. O S T E O I N D U C T I O N
• Materials That Have The Capacity To Induce Bone Formation When Placed Into A Site Where No Bone Formation Will
Otherwise Occur Are Termed Osteoinductive.
• These Materials Recruit Mesenchymal Stem Cells Or Their Progeny To Infiltrate The Material Of Tissues
(Chemoattraction And Migration), And Then They Induce Multipotential Cells To Multiply And Become Cells That
Make Up The Regenerating Bony Callus (Proliferation And Differentiation)
• The Best Known Example Of Osteoinductive Materials Is Demineralized Bone Matrix.
• By Decalcifying Bone (Usually Allogeneic) In Processes That Do Not Inactivate The Biologically Active Components
Of Bone, Organic Matrix Is Exposed, Along With A Plethora Of Bone Stimulatory Cytokines Strapped Within The
Organic Matrix During Bone Formation.
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10. O S T E O C O N D U C T I O N
• Material That Provides A Scaffold For Mesenchymal Stem Cells And Their Progeny To Migrate Into, And Proliferate
With, Is Termed Osteoconductive
• These Physical Materials (Biologic Or Synthetic) Of Three-dimensional Shape Offer Appropriate Framework Surface
Characteristics For Adherence Of Mesenchymal Stem Cells, Osteoblasts, Osteocytes, Chondroblasts, And Chondrocytes,
And Interconnecting Porosity For Cellular Proliferation And, Most Important, Vascular Ingrowth
• Such Materials May Or May Not Impart Load-bearing Characteristics During Bone Regeneration, May Or May Not Be
Absorbable, And May Be Naturally Occurring (E.G., Trabecular Matrix Of Cancellous Bone) Or Synthetic (E.G., Porous
Bioceramics)
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11. O S T E O P R O M O T I O N
• A Material Or Physical Impetus That Results In Enhancement Of Regenerating Bone Is Termed Osteopromotive.
• Examples Of Osteopromotive Substances Are Platelet-rich Plasma, Hydrogels, And Biphasic Calcium Phosphate
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12. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
• As Mentioned Previously, Autogenous Cancellous Bone Grafts Are Still Recognized As A Clinical Gold Standard For
Enhancing Bone Formation And Remain The Mainstay Of Bone Grafting, Especially In Veterinary Medicine.
• This Form Of Bone Graft, Taken From The Host And Usually At The Time Of Surgery, Will Institute All Of The
Strategies To Enhance Bone Regeneration.
• The Trabeculae Are Lined With Osteoblasts That Provide Osteogenesis Under The Influence Of Local Cytokines.
• The Disrupted Bone Matrix Of The Cancellous Bone Releases An Entire Milieu Of Cytokines And Growth Factors
From The Extracellular Matrix; These Substances Are Osteoinductive
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13. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
• A Properly Harvested Graft Maintains A Structural Scaffold That Serves As A Special Conduit On Which New Bone
Can Form (I.E., Osteoconduction)
• Hemorrhage And The Resulting Clot Contain Activated Platelets, And Hence Growth Factors From The Released
Platelet Alpha Granules, Which Contain IGF-1, PDGF, And Tgf-β, Serving As An Osteopromotive Function
• Autogenous Cancellous Bone Collection Is Well Tolerated In Veterinary Patients, Unlike In Human Beings, For Whom
Pain At The Donor Site Can Persist For Months Or Years.
• The Most Common Donor Sites Are The Proximal Aspect Of The Humerus And The Wing Of The Ilium.
• Common, But Less Popular, Donor Sites Include The Proximomedial Region Of The Tibia, The Subtrochanteric Region
Of The Femur, And The Condyles Of The Femur.
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14. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
• Other Sites Used Less Frequently Include The Caudoventral Portion Of The Mandible (For Periodontal Surgery) And
The Rib.
• Complications Associated With Harvesting Of Cancellous Bone Graft Are Rare In Dogs, But Fracture And Premature
Growth Plate Closure Have Been Reported.
• Restoration Of Cancellous Bone At The Donor Site Has Been Evaluated.
• Restoration Occurs More Completely In The Proximal Region Of The Humerus Than In The Proximal Region Of The
Tibia, And It Is Sufficient For Repeat Grafting By 8 Weeks In The Proximal Humerus And By 12 Weeks In The
Proximal Region Of The Tibia
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15. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
• The Optimal Size Of The Cancellous Particles Is Between 3 And 6 Mm
• Viable Cell Numbers Will Sharply Decline Very Rapidly, Even Under Optimal Storage Conditions
• If The Graft Is To Be Stored For Any Length Of Time, It Should Be Placed In Blood Or Saline Because Drying Will
Significantly Lower Cell Viability In As Little As 2 Hours And Cell Numbers In As Little As 2 To 3 Hours
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16. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
Harvesting of an autogenous cancellous bone graft using two
orthogonally placed Gelpi retractors to expose the cis-cortex for
intramedullary pin penetration of the metaphyseal bone.
Brun bone curette used to harvest
autogenous cancellous bone.
Autogenous cancellous bone graft harvesting technique.
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17. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
Receptacles for holding autogenous bone graft.
Segments of cancellous bone are removed
from the proximal part of the humerus or
ilium and placed in the barrel of a syringe.
This keeps the graft moist, and as the blood
component clots the graft assumes the shape
of a wedge that is convenient for placing in
the area needed
The walls and bottom of a sterile receptacle
are moistened with a saline-soaked sponge in
preparation for temporary storage of
harvested cancellous autograft.
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18. A U TO G E N O U S C A N C E L L O U S B O N E G R A F T S
Receptacles for holding autogenous bone graft.
The harvested cancellous
autograft remains in the sterile
receptacle until transplantation
is performed.
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19. HEALING OF AUTOGENOUS CANCELLOUS BONE GRAFTS
• An Autogenous Cancellous Bone Graft Is Incorporated Into A Fracture Site In Several Stages.
• The Initial Fracture Hematoma Becomes Gradually More Organized And Fibrotic Over Time, And It Is Resorbed
Within The First 1 To 2 Weeks.
• Within Minutes To Hours, An Inflammatory Response Attracts Cells, Including Lymphocytes, Plasma Cells, And
Mononuclear Cells. Revascularization And Osteoinduction Begin, And Within 5 Days Of Grafting, Capillary Loops
Enter The Connective Tissue Within The Graft.
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20. HEALING OF AUTOGENOUS CANCELLOUS BONE GRAFTS
• There Is A Gradual Increase In The Number Of Blood Vessels During The Next 10 Days.
• Necrotic Tissue Is Resorbed And The Graft Is Fully Vascularized By 20 Days.
• Woven Bone Will Initially Be Deposited On The Necrotic Trabeculae Of The Graft And Will Later Be Remodeled Into
Lamellar Bone.
• Over Several Months, Remodeling Of The Lamellar Bone Elements Will Lead To A New And Continuous Cortical
Surface (Corticalization).
• Deeper Trabecular Bone Will Continue To Develop Along With Hematopoietic Elements (Medulization).
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21. HEALING OF AUTOGENOUS CANCELLOUS BONE GRAFTS
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Woven Bone Lamellar Bone

22. ALLOGRAFT-BASED BONE GRAFT SUBSTITUTES
• Allogeneic Bone Has Both Osteoinductive And Osteoconductive Properties And Capabilities But Lacks The Osteogenic
Cells Present In Fresh Autogenous Bone Grafts.
• Due To Its Naturally Occurring Bone Morphogenetic Proteins, Exposed Through Demineralization, Allograft Can
Facilitate Induction Of Bone Growth Through These Same Mechanisms.
• In Its Cancellous Form, Allograft Provides An Osteoconductive Scaffold For Vascular Ingrowth And For Osteoblast
Migration
• Unlike Autografts, Allograft Volume Or Size Is Not Limited By What Can Be Safely Harvested From The Patient, Nor
Does Allograft Bone Have The Associated Morbidity Risks Involved With Autograft.
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23. ALLOGRAFT-BASED BONE GRAFT SUBSTITUTES
• It Does, However, Have Some Risks Of Its Own, Including A Small Risk Of Disease Transmission And Remote
Concerns About Histocompatibility Issues.
• Histocompatibility Issues Are Ameliorated By Most Processing Methods For Bone Grafts Which Include Scraping Of
Periosteum, Purging Of Marrow Elements Such As Lipids, Freezethawing, Freeze-drying, And Treatment With Acids
And Other Agents, As Well As Irradiation.
• Transplanting Bone That Is Fresh And Unprocessed Stimulates A Significant Immune Response In The Host.
• No Cases Of Disease Transmission Using Processed (E.G., Freeze-dried And/Or Demineralized) Bone Products Have
Been Reported During Approximately The Past 30 Years.
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24. ALLOGRAFT-BASED BONE GRAFT SUBSTITUTES
• Since 1996, When Commercially Available Allograft Bone Was First Offered For Veterinary Use, There Have Been No
Reports Of Disease Transmission From Allograft To Recipient.
• Frozen Grafts Are Preserved At A Storage Temperature Of −70°c.
• Freezedried Grafts May Be Irradiated With Gamma Irradiation To Permit Room Temperature Storage.
• Irradiation Has Bactericidal And Virucidal Effects, And At Relatively Low Doses, It Has Been Shown To Provide
Virtually Complete Sterility.
• It Is Clear That High Doses Of Radiation Can Have Deleterious Effects On Osteoinductivity And On The
Biomechanical Properties Of Load-bearing Bone.
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25. ALLOGRAFT-BASED BONE GRAFT SUBSTITUTES
Fusion of the cervical vertebral
column at C5-C6 and C6-C7, using
interbody cortical allografts and
autogenous cancellous bone. (Image
courtesy Dr. Peter Early, North
Carolina State University.)
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26. HEALING OF CORTICALALLOGRAFTS
• Cortical Allografts Are Used In Situations In Which Structural Support Is Needed, And In Which Large Segments Of
Lost Cortical Bone Need To Be Replaced.
• These Grafts Have Been Used For Replacement Of Diaphyseal Bone Defects Due To High Degrees Of Comminution.
• Because Of The Slow Rate Of Healing Of Cortical Allografts, More Biologic Methods Are Currently Employed.
• Cortical Allografts Are Used To Replace Segments Of Bone That Are Removed In Association With Limbsparing
Procedures For The Surgical Management Of Osteosarcoma And Other Bone Neoplasms
• In These Situations, The Allograft Serves Primarily A Mechanical Function, Rather Than A Regenerative Function,
Because The Healing Process Far Exceeds The Life Expectancy Of The Patient.
• Cortical Ring Allografts Are Also Used As Intervertebral Body Spacers In Spinal Fusion Applications.
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27. HEALING OF CORTICALALLOGRAFTS
• Cortical Allografts Heal In A Different Manner Than Cortical Autografts.
• Cortical Allografts Heal Much More Slowly And With A Much Greater Inflammatory Response Caused By Residual
Antigenic Material
• Serving Mainly In A Structural Support Function, Allografts Do Not Supply Osteogenic Cells And Are Not Likely To
Supply Clinically Significant Amounts Of Osteoinductive Elements.
• For Cortical Allografts To Provide Structural Assistance, They Must Be Stabilized By Rigid Internal Fixation And
Interfragmentary Compression.
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28. DEMINERALIZED BONE MATRIX
• Bone Allograft Is Used In Nearly 1.5 Million Human Orthopedic And Dental Surgeries Per Year; Most Contain
Demineralized Bone Matrix
• Demineralized Bone Matrix Is Bone That Has Been Ground To Specific Particle Sizes And Has Been Decalcified With
The Use Of Acids (Typically Hydrochloric Acid).
• Canine Bone Consists Of Approximately 22% To 25% Calcium Before Demineralization, And After A Typical
Demineralization Procedure, This Is Reduced To <3%.
• It Can Subsequently Be Preserved By Freezing Or Freeze-drying.
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29. DEMINERALIZED BONE MATRIX
• Demineralized Bone Matrix Has Been Shown To Be Effective In Bone Healing And Can Be Packed Easily Into Bone
Voids, Mixed With Carriers Such As Putties, Or Mixed With Autograft, Exogenous Recombinant Bone-specific Growth
Factors, Or Other Growth Factors Such As Platelet-derived Growth Factors; It Can Also Be Combined With Stem Cells.
• Demineralized Bone Matrix Mixed With Allogenic Cancellous Bone Chips Provides A Combination Of Osteoinductive
Bone Matrix And An Osteoconductive Scaffold For Vascular Invasion And Osteoblast Migration Into The Site
• The Porous Nature Of Cancellous Bone Provides A Scaffold.
• The Advantage Of Allogenic Cancellous Chips As A Structural Matrix Is That They Are Resorbed By The Body Over
Time As Part Of The Normal Metabolic Process And Are Replaced By The Patient’s Own Bone
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30. CELL-BASED STRATEGIES FOR BONE REGENERATION
• M e s e n c h y m a l S t e m C e l l s
BONE MORPHOGENETIC PROTEINS FOR AUGMENTATION OF BONE REGENERATION
 Although Many Cytokines Have Been Known To Encourage Bone Formation, Only BMP-2 And BMP-7
Are Commercially Available For Clinical Use
SYNTHETIC MATERIALS FOR BONE GRAFT SUBSTITUTES
 Ceramics
 Calcium Phosphate Ceramics
 Coralline Bone Graft Substitutes
 Tricalcium Phosphate
 Biphasic Calcium Phosphate
 Nanocrystalline Calcium Phosphate
 Calcium Sulfate
 Hydrogels
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31. Thanks For Your Attention