The document discusses bone tissue engineering and the challenges in developing bioartificial bone. It covers the composition of natural bone, current treatments for bone defects, strategies for bone tissue engineering including the use of scaffolds, cells, growth factors and bioreactors. Key challenges discussed are achieving timely angiogenesis within scaffolds and developing biomaterials that degrade predictably and cause minimal immune response.

3.  Introduction
 Bioartifical bone
 Scaffold
Classification
Techniques
 Bioreactors
 Vascularization
 Challenges
 Conclusion

4. Mineral phase 70%
Hydroxyapatite 95%
Other components 5%(Mg, Na, K, F, Zn,
Sr, C)
Organic phase 30%
Bone matrix 98%
(Collagen 95%,
Non-collagenous proteins 5%)
(BMPs, TGF)
Bone cells 2%
(Osteoblasts,Osteocytes,Osteoclasts)

5.  Highly efficient and tightly regulated process
 Result of continuous interplay between growth factors and
cytokines for both initiation and regulation of remodeling
process
 Healing of fractures- standard conservative or surgical therapy
 Extended bone defects - more sophisticated treatment
 Bone reconstruction - Bone grafting procedures, segmental
bone transport, distraction osteogenesis or application of
biomaterials

6.  Current gold standard treatment of critical sized
bone defects - autologous bone grafting
 Advantages of utilizing synthetic bone scaffolds
include
• Elimination of disease transmission risk
• Fewer surgical procedures
• Reduced risk of immunogenicity
• Abundant availability

7.  “Tissue engineering is an interdisciplinary field that
applies the principles of engineering and life sciences
toward the development of biological substitutes that
restore, maintain, or improve tissue function.”
(Langer and Vacanti, 1993)
 Tissue engineering ~ Regenerative medicine

8.  The intended clinical use defines the desired properties of
engineered bone substitutes
 Initial vascularization - essential for enhanced engraftment
and prevention of infection
 Mechanical stability
 Osteoconductivity
 Osteoinductivity
 Osteogenicity
 Ease of handling

9.  Multiple bone tissue engineering strategies –
cell transplantation, acellular scaffolds, gene therapy and growth factor
delivery
 Two primary tissue engineering strategies – emerged as most
promising approaches
› Before implantation, isolation of MSCs – in vivo expansion – seeding
on to a synthetic scaffold- allow to produce ECM on scaffold-
implantation into osseous defect
› Implantation of an acellular scaffold immediately after injury/ bone
removal

10.  Scaffolds
 Osteogenic cells
 Osteoinductive factors
Bioreactors

11.  Artificial matrices designed to mimic the
mechanical and biological properties of the
tissue matrix (Pou, 2003)
 Provides three-dimensional spaces for the cells
to undergo proliferation and differentiation
 Serves as carriers to transfer cells and bioactive
materials to defect sites

12. 1. Mechanical strength
2. Osteoconductivity
3. Osteoinductivity
4. Porous structure (pore numbers, size and morphology)
Pore sizes <15-50µm result in fibrovascular ingrowth, 50-150µm -
osteoid formation and >150µm - mineralized bone
5. High surface/volume ratio
6. Elicitation of minimal immune responses and inflammation
7. Biodegradability (Degradation rate: formation rate of new tissue)
8. Capable of sterilization without loss of bioactivity
(Khang et al., 2006; Salgado et al., 2004)

13. CERAMIC
• Hydroxy apatite
• Tricalcium
phosphate
• Coral
exoskeleton
NATURAL
• Cellulose
• Starch
• Chitin
• GAG
• Hyaluronic acids
& proteins
SYNTHETIC
• PLA
• PLGA
• PLLA
• PGA
• POLYETHYLENE
GLYCOL
HYDROGEL
• chitosan
• alginate
• silk fibroin

14.  Constitute main part of natural bone matrix
 Synthetic calcium based ceramics
(Hydroxy apatite, tricalcium phosphate ceramics)
• Very fragile
• Low mechanical stability
• Degradation rate not predictable
 Natural ceramic organic composites (coral
exoskeleton)
• Best mechanical properties of porous
calcium based ceramics
• Interconnected porous architecture ~
spongy bone

15.  Low immunogenecity
 High inherent bioactive properties
 Capacity for good interaction with
host tissue
 Unlimited sources
 High chemical diversity
 Inhibitory effects on bacterial
growth
(Baldwin and Saltzman, 1998)

16. Collagen scaffold
 Easily obtainable from tissues
 Controlled degradation rate
 Ideal carrier for bone morphogenic
protein
 Increases adhesion and maturation of
osteogenic cells
 Poor mechanical properties

17.  Most frequently used
 Biodegradable
 Easily prepared in different
shapes and dimensions
 Superior mechanical properties
 Appropriate degradation rate
 Good microstructure
 Synthetic scaffolds with ceramic
nanoparticles – revolutionary
approach
PLLA SCAFFOLD
PGA SCAFFOLD

18.  Consists of a cross linked polymer
network inflated with solvent such as
water
 Ability to reversibly swell or shrink (up to
1000 times in volume) due to small
changes in their environment (pH,
temperature, electric field)
 Can deliver sizeable stress
 Polyvinyl alcohol (PVA), polyacrylic acid
(PAA), polyacrylonitrile (PAN)

19.  Merits of injectable polymer gels over preformed
scaffolds
• Minimally invasive implantation
• Ability to fill desired shape
• Easy incorporation of various therapeutic agents
 Demerit
• Strength and structural limitations

20.  Cells trapped within hydrogel
during gelation process
 Alginate & silk fibroin –
• Good biocompatibility
• Flexibility
• Mechanical stability
 Multilayer hydrogels– appropriate
scaffolds for cell co- culture
(Rowley et al., 1999)

21.  Fiber bonding
 Solvent casting
 Particulate leaching
 3 D prototyping allows
• Completely interconnected pore network
• Highly regular & reproducible morphology
• Microstructure which varies across scaffold matrix
• Solvent free production
• Ultra- thin bioabsorbable membrane with nano-porosity

22. FUSED DEPOSITION MODELLING
BIOPLOTTER
STEREOLITHOGRAPHY

23.  Transplantation along with the appropriate scaffolds into the
bone defect or attraction from the host by osteoinductive
factors
 Essential characteristics
• Isolation and expansion efficiency
• Stability of osteoblastic phenotype
• In vivo bone formation capacity
• Long term safety
 Mesenchymal stromal cells, bone marrow stromal cells, periosteal
cells, osteoblasts

24.  Applied as crude and hardly standardized mixture of
proteins or as isolated factors
 Augment osteogenic capacity of tissue engineered bone
constructs
 Modulate proliferation and differentiation of implanted
osteogenic cells
 Attract precursor cells from host to invade scaffolds and
induce osteoblastic differentiation
 Bone morphogenic proteins (BMPs) - most potent
osteoinductive factors (BMP2, BMP4, BMP7)
(Wozney and Rosen, 1998)

25.  Requires optimized
pharmacokinetics
 Direct injection not effective
 Controlled release via carrier
like polymer hydrogel
PROTEINS COMMON
NAME
BMP-2 bmp2a
BMP-3 osteogenin
BMP-4 bmp2b
BMP-6 Vgr-1
BMP-7 Op-1
BMP-13 Cdmp-2, gdf-6
BMP-14 Cdmp-1, gdf-5

26.  Previously , static cell culture – to establish tissue
engineering culture
 Demerits of static cell culture
• Improper diffusion of nutrients to deeper parts of the
scaffold
• Uneven distribution of cells throughout the scaffolds
• Unable to provide mechanical stimulation

27.  Objectives
• To enhance in vitro performance of
osteogenic cells before implantation
• To provide uniform distribution of
cells within scaffolds
• To provide cells deeper in the
scaffold with sufficient nutrients
• To expose the cells to mechanical
stimulation
a) Spinner flasks
b) Rotating wall vessels
c) Direct perfusion bioreactors
d) Static

28. Perfused bioreactor system

29. 1. Placing the cells suspended in medium as a drop on the top
of surface of the scaffold
2. Immersion of the scaffold
3. Injection of cells and medium
4. Agitation of cells and scaffold complex using an orbital
shaker
5. Loading the cells by perfusion using an appropriate
bioreactor
6. Using centrifuge
7. Use of nanoparticles and magnetic forces

30. Scaffold seeding method driven by surface acoustic waves

31.  Methods of Administration of GFs &
other bioactive molecules to promote
bone formation and repair
• Bolus injection
• Surface adsorbed protein release
• Osmotic pumps
• Controlled release from
biodegradable scaffolds
 Biodegradable bone scaffolds capable
of sustained and controlled drug
elusion- ideal candidates

32.  Drawbacks of Conventional systemic delivery of
antibiotics
• Systemic toxicity
• Renal, liver complications
• Poor penetration into necrotic tissue
• Need for hospitalization
 Most common biodegradable polymer/antibiotic
combination – PLGA scaffolds loaded with
antibiotics such as ciprofloxacin, gentamicin,
vancomycin
 Antibiotic stability within scaffold, antibiotic
deactivation during fabrication

33.  Prerequisite for formation of high
quality bone
 In vitro cellular constructs - in vivo
(interstitial fluid diffusion and blood
perfusion)
 Diffusion only provide support to
cells within maximum range of 200
µm into matrix

34. 1. Angioinductive growth factors
2. Endothelial cells
3. Local factors at recipient site
4. Surgical angiogenesis – Intrinsic mode
Extrinsic mode of
neovascularization

36.  Work together in highly co-ordinated manner to
induce endothelial cell outgrowth and formation of
functional vessels
 Promising tools for induction and acceleration of
vascularization processes in 3-D scaffolds
 VEGF and bFGF
 Immobilization of angiogenetic growth factors allows
for optimised release kinetics and longer lasting
effects (fibrin gels or heparin binding release systems)

37.  Derived from microvasculature,
umbilical veins or large blood vessels
 Form networks of capillaries and gain
access to the recipient’s circulation
3. Local factors at recipient
site
 Superior quality of tissue
 Appropriate local environment
(bacterial load, chronic scarring)
 Tissue constructs s/be sufficiently thin

38.  Neovascular bed originates from periphery of
construct –implanted into site of high
vascularization
 Subcutaneous, intramuscular, intraperitoneal
implantation
 Generation of axially vascularized tissues
 Techniques for induction of axial
vascularization
1. Flap prefabrication
2. “Free style” vascularization

39.  Creation of prefabricated composite flap according to
complex geometry of the defect
 Revascularization phenomenon directly related to host tissue
vascularity
 Two step process
1. Formation of tissue component in desired shape –
implantation into region with a vascular axis suitable for
microsurgical transfer
2. Harvesting autologous implant en bloc with surrounding
tissue and vascular pedicle as free flap

40.  Acquisition of vascularization from the tissue block by
an implant - Connection of flap to local circulation by
means of microvascular anastomosis
 Two strategies for flap prefabrication
• Wrapping of bone graft in axially vascularized
tissues
• Implantation of vascular axis into bone graft

41.  Intrinsic mode of vasculrization – construct
acquires an inherent perfusion- does not have to
depend on favorable local conditions (achieved
by inducing angiogenesis from centrally located
vascular axis)
 Allows generation of new types of flaps
independent of the vascular anatomy of bone
transplant

43.  Creation of vascular axis using
vein grafts- generation of
vascularized bone units relatively
independent of anatomical
limitations
 Vascularization using A-V loop
(arteriovenous vessel loop)
• More than 90% vascularization of
scaffold
• Less pronounced inflammatory
reaction
• Significant increase in number of
initially engrafted osteoblasts

45. Approaches in vascularizing engineered bone scaffolds

46.  Chitosan sponges incorporated with PDGF by simple
adsorption- significant regenerative capacity in craniotomy
defect – Park et al., 2000
 Combination of coral, a natural calcium carbonate- based
ceramic and MSC for reconstruction of large bone defects -
Petite et al., 2000
 Enhanced bone formation was observed in critical sized
cranial defect model in rats when genetically modified cells
expressing BMP-2 seeded in titanium fiber meshes were
implanted (Blum et al., 2003)
 BMP-2 releasing PLA scaffolds induced bone formation pon
implantation into subcutaneous dorsum – Yang et al., 2004

47.  Hou et al., 2006 evaluated autologous bone marrow derived
MSCs seeded corals with added rhBMP2 in reconstruction of
New Zealand rabbit calvarial bone defects
 Marcacci et al., 2007 reported use of bone marrow derived
MSCs implanted on macroporous HA scaffold in four
patients with large bone defects

48.  Angiogenesis in a timely fashion within the scaffold
construct
 New biomaterials causing minimal foreign body
response & degrading in completely predictable fashion
 Readily available, safe, off-the-shelf supplies of
osteogenic cells
 Advanced manufacturing systems are required that can
fabricate complex scaffolds with spatially controlled
distributions of materials, microstructures, cells and
growth factors

49.  Tissue engineering has tremendous potential to
overcome limitations of the existing therapies for
bone replacements
 Looking into the progress made in field of bone
tissue engineering in recent past, the time not far
when ready to use customized bone substitutes will
be available in the market for day to day clinical
practice.