2. Why COLLAGEN AND HYDROXYAPATITE
 Collagen is used extensively as a scaffold
biomaterial due to its biocompatible and
biodegradable properties .
 Indeed, both collagen type I and
Hydroxyapatite were found to enhance
osteoblast differentiation but when
combined they showed osteogenesis
 From an orthopedic perspective however,
collagen scaffolds are limited by their poor
mechanical characteristics and for this
reason we aim to combine collagen with
Hydroxyapatite to improve their mechanical
properties
 Also, if alone collagen is used , it is
degraded by the collagenase of the body.
Thus, to decrease the rate of degradation
also Coll/HA composites are in demand Eucare pharmaceuticals Ltd. Sybograf-C
http://www.eucareindia.com/product.html

3. The natural bones contain mainly
collagen and Hydroxyapatite. That is the
reason because many researchers try
for (nano)Hydroxyapatite/collagen
composite for hard tissue repairing
Composition of Bone Tissue
• 25% Water
• 25% Protein or organic matrix
95% Collagen Fibers
5% Chondroitin Sulfate
• 50% Crystallized Mineral Salts
Hydroxyapatite
(Calcium Phosphate)

4. Coll/HA composite
 (Left) Fiberized hydroxyapatite and collagen compound composite
immediately after adjustment
 (Right) Hydroxyapatite and collagen compound composite made
porous

5. Advantages/Properties of Coll/HA
composites
 Biodegradable Resorbed into the body over time
 Biocompatible and Bioactive Facilitates complete osteogenesis
 High porosity Facilitates bone cell migration through matrix
Increases cellular nutrient and waste exchange
 High permeability Facilitates conductivity of fluids through matrix
 Mechanically strong Facilitates handling and ease of use
Provides structure within which bone can reform
 High degree of pore connectivity prevents avascular necrosis , increases cell
mobility
 The interest in temporary substitutes is that they permit a mechanical support until
the tissue has regenerated and remodeled itself naturally.
 Furthermore they can be seeded with specific cells and signaling molecules (growth
factors,VEGF, TGR) in order to maximize tissue growth and the rate of degradation
and absorption of these implants by the body can be controlled.

6. Preparation of microspheres
Water-in-Oil emulsion system (microspheres of 200- 300 µm)
 Bovine collagen in phosphate-buffered saline (PBS) was mixed with HA
powders in a ratio of 35:65 (w/w, collagen:HA) at 4°C, the mixture was
added into olive oil while stirring at 400 rpm at 37°C for 2 h to allow the
reconstitution of collagen.
 Glutaraldehyde was then added to the emulsion at a final concentration
of 2.5% in the aqueous droplets, and the mixture was incubated for 1 h.
After centrifugation, collagen/HA microspheres were collected from the
lower portion of the tube and washed repeatedly with 0.1m PBS.
 Microspheres in suspended form can be delivered to the targeted and
hard-to-reach areas with ease,e.g. by syringe injection for applications
as cell or tissue carriers, bone grafting and drug delivery and there is no
need of surgery in this case , They can be directly injected into the bone
defect

7. Freeze drying process
(lyophilization)
 Firstly a stable nHA suspension was produced and added to a collagen
slurry (suspension method), and secondly, porous collagen scaffolds were
immersed in nHA suspension after freeze-drying (immersion method).
 Significantly stronger constructs were produced using both methods
compared to collagen only scaffolds, with a high porosity maintained
(>98.9%).
 It was found that Coll-nHA composite scaffolds produced by the suspension
method were up to 18 times stiffer than the collagen control .
 The suspension method was also more reproducible, and the quantity of
nHA incorporated could be varied with greater ease than with the immersion
technique. In addition, Coll-nHA composites display excellent biological
activity, demonstrating their potential as bone graft substitutes in
orthopaedic regenerative medicine.

8. Effect of pore size
 Pore size is an essential consideration in
the development of scaffolds for tissue-
engineering
 If pores are too small cell migration is
limited, resulting in the formation of a
cellular capsule around the edges of the
scaffold. This in turn can limit diffusion of
nutrients and removal of waste resulting in
necrotic regions within the construct.
 Conversely if pores are too large there is a
decrease in surface area limiting cell
adhesion.
 Also, large pore size may compromise the
mechanical properties of the scaffolds by
increasing void volume
 Pores greater than ~300 µm lead to direct
osteogenesis while pores smaller than
~300 µm can encourage osteochondral
ossification

9. Effect of scaffold pore size
 Previous studies in bone tissue-engineering have indicated a range
of mean pore sizes (96–150 µm) to facilitate optimal attachment.
 Other studies have shown a need for large pores (300–800 µm) for
successful bone growth in scaffolds. These conflicting results
indicate that a balance must be established between obtaining
optimal cell attachment and facilitating bone growth.
 Cell adhesion decreased with increasing pore size and that the
highest levels of cell attachment were found on the scaffolds with
the smallest pore size (96 µm)
 Initial studies demonstrated that the minimum pore size for
significant bone growth is 75–100 µm with an optimal range of 100–
135 µm.Since this early work it has been reported that pores
greater than ∼300 µm are essential for vascularisation of constructs
and bone growth, while pores smaller than ∼300 µm can encourage
osteochondral ossification.

10. Col/HA Products in market
Product/ Company Name
 HydroxyColl
www.enterprise-ireland.com/en/Events/.../Poster-HydroxyColl.pdf
 Ossfill
SEWON CELLONTECH CO., LTD.
http://www.gobizkorea.com/blog/ProductView.do?blogId=cellontech&i
d=982532
 SyboGraf™- C
Eucare Pharmaceuticals Private Limited
http://www.eucareindia.com/product.html
 Collapat® II
http://www.biomet.fi/ammattilaiset/biomateriaalit/luunkorvikkeet/collap
at
 MCH-Cal™ : <850 micron, < 250 micron and < 150 micron
Waitaki biosciences
http://www.waitakibio.com/manufacturer/natural-calcium

11. Other Competitive Three dimensional
porous biomaterials
Collagen / Hydroxyapatite/ Tricalcium phosphate
 Cross.Bone® Matrix is made of hydroxyapatite (HAP), β-
tricalcium phosphate (β-TCP) and collagen. The biphasic and
synthetic bone substitute has an optimized micro and macro-
porosity. http://www.implants.fr/en/pageLibre0001164f.asp
 Nano-carbonated Hydroxyapatite/Collagen/PLGA
(Poly glycolic acid)or poly(lactic)-co-glycolic acid
 HAC-PLA scaffolds
 nano-Hydroxyapatite/ Collagen/Calcium alginate
 Collagen /Calcium alginate Fibracol http://skin-wound-
care.medical-supplies-equipment-company.com/product/fibracol-
plus-collagen-wound-dressing-with-alginate_5049.html

12. Other competitive composites
available in market

13. References
 Karageorgiou V. Kaplan D. Porosity of 3D biomaterial scaffolds and osteogenesis.Biomaterials
2005;26:5474-91.
 DA Wahl et al. Collagen-Hydroxyapatite Composites for Hard Tissue Repair.European cells and materials
Vol. 11. 2006 (43-56)
 Murphy CM, Haugh MG, O'Brien FJ. The effect of mean pore size on cell attachment, proliferation and
migration in collagenglycosaminoglycan scaffolds for tissue engineering. Biomaterials. 2010;31(3):461-6.
 Kuboki Y, Jin QTakita H. Geometry of carriers controlling phenotypic expression
in BMP-induced osteogenesis and chondrogenesis. J Bone Joint Surg Am2001;83-A Suppl 1:S105-15.
 Cunniffe GM, Dickson GR, Partap S, Stanton KT, O'Brien, FJ. Development and characterisation a collagen
nano-hydroxyapatite composite scaffold for bone tissue engineering. Journal of Materials Science: Materials
in Medicine. 2010;21(8):2293-8.
 Hsu F-Y, Chueh S-C, Wang JY (1999) Microspheresof hydroxyapatite/reconstituted collagen as supports for
osteoblast cell growth. Biomaterials 20: 1931-1936
 Ciara M Murphy1 and Fergal J O'Brien1,2†Understanding the effect of mean pore size on cell activity in
collagen-glycosaminoglycan scaffolds. Cell Adh Migr. 2010 Jul-Sep; 4(3): 377–381
 Wu et al, Studies on the microspheres comprised of reconstituted collagen and hydroxyapatite. Biomaterials
25 (2004) 651–658
 J.-S. SUN ET AL ,Collagen-Hydroxyapatite/Tricalcium Phosphate Microspheres as a Delivery System for
Recombinant Human Transforming Growth Factor-b 1,International Society for Artificial Organs,
2003,27(7):605–612

14. Conclusion
 Collagen along with Hydroxyapatite is the best choice for
artificial bone , or as a filler in bone defects, due to its quick
adaptation in the body.
 For preparation of the scaffold many methods are available ,
among them the best is the freeze drying process in which
the nHA is combined with collagen and then freeze dried.
 The pore size of this product should be decided on the basis
of the exact use of the product for eg:if Osteogenesis is the
aim , we should use 300-800 micron pore size.
 And , if the choice is only cell attachment and osteochondal
ossification we should use less than 300micron pore size .
 Also, from the market study , we found that there are handfull
of products containing the combination of Collagen and
hydroxyapatite.