Collagen application

1. CURRENT
PROJECTS
Presented By: Akshita Vyas

2. SCENARIO OF WOUND CARE MANAGEMENT IN
INDIA
 In India alone , there are about 35 million diabetics -
the figure is expected to rise to 52 million by 2025
 According to The Hindu (Online edition of India's
National Newspaper, Wednesday, Apr 05, 2006), Every
year more than 40,000 diabetic patients undergo leg
amputations in India
 According to WHO, every 30 second an amputation
takes place in India due to diabetes. In fact, WHO has
declared India the 'diabetic capital of the world'
 In fact, Patients with pressure ulcers, diabetic ulcers,
and traumatic wounds represent major market
segments in India. Projections indicate that these
patient populations continue to increase rapidly in the
next century.
 Thus there is a need , with increasing realization of the
value of advanced wound care.

3. FOR DIABETIC FOOT ULCERS WE REQUIRE A
MORE ADVANCED WOUND CARE PRODUCT
THE ANSWER TO IT IS SILVER NANO PARTICLES
 It is proven fact that AgNP have
properties like good conductivity,
chemical stability, catalytic , antibacterial
activity, antifungal, anti-
viral,antiinflammatory,so they have been
used in various medical devices.
(Mukherjee et al., 2001; Sondi and
Branka, 2004; Chen and Schluesener,
2008)
 It has been found that smaller
particles generally present an enhanced
antibacterial activity if compared with
their larger counterparts.
 The range of the size other medical
devices uses is 10-100 nm
(20,40,60,80,100nm)

4. SILVER NANO PARTICLES(SNP’S):EC50
,IC50
(EFFECTIVE CONCENTRATION)
(INHIBITORY CONCENTRATION)
 Also, we can measure the effect of
silver Nano particles on the
growth of the cells.
 In this way , we can measure the
effective concentration of the
silver Nano particles to be added
in this scaffold.
 By performing cell-based cytotoxic
assay we can measure the EC 50
or IC 50 to see the concentration
of the SNPs to be used for the
good growth of the cells.
 This assay can be performed on a
normal 96well plate with definite
cell volume and varying
concentration of SNP’s

5.  Silver nano powder
 Nanoshel LLC - Intelligent Materials Pvt Ltd

6. COMPETITIVE PRODUCTS
Puracol Plus AG+ Wound (COLLAGEN & SILVER
Dressing with NANO)
Antibacterial Silver
Smith and Nephew Colactive Ag
Collagen Dressing
Eucarepharmaceuticals
Collagen Film With
SORE-TREAT-SLR – Silver Sulphadiazine SilvaKollagen Gel
Helix pharma Neuskin-FS Silver Antimicrobial
(Collagen Based Cream Collagen
of Silversulfadiazine) .

7. NEW INSIGHT IN TO
BONE DEFECTS
Collagen Hydroxyapatite scaffold

8. 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

9. WHY COLLAGEN AND
HYDROXYAPATITE ?
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

10. ADVANTAGES OF COLLAGEN /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 careform
 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.

11. COLLAGEN /HA COMPOSITE
 (Left) Fiberized hydroxyapatite and collagen compound composite
immediately after adjustment
 (Right) Hydroxyapatite and collagen compound composite made
porous

12. Collagen /Hydroxyapatite scaffolds
Work going on . . . . .
 Collagen/Hydroxylapatite Scaffolds protocol
 Standardizing the protocol on small scale
 Studying the Detailed Chemistry of the pore size
formed in this scaffold
 Studying the efficacy of the formed scaffold

13. HYDROXYLAPATITE
 Chemical formula Ca5(PO4)3OH
 CAS No 12167-74-7
 SYNONYMS
1. PENTA-CALCIUM HYDROXIDE
TRIPHOSPHATE
2. TERT-CALCIUM PHOSPHATE
3. TRI-CALCIUM DI-ORTHOPHOSPHATE
4. TRI-CALCIUM (ORTHO)PHOSPHATE
5. TRI-CALCIUM PHOSPHATE
6. TRI-CALCIUM PHOSPHATE (BETA-FORM)
7. CELLULOSE HYDROXYAPATITE
8. CALCIUM HYDROXIDE PHOSPHATE
9. CALCIUM PHOSPHATE TRIBASIC
10. CALCIUM ORTHOPHOSPHATE;
11. BETA-CALCIUM PHOSPHATE TRIBASIC
12. BETA-TRICALCIUM PHOSPHATE

14. PROTOCOL FOR THE SCAFFOLD
PREPARATION: SLURRY
PREPARATION
 Prepare a uniform slurry of Collagen and
Hydroxylapatite
 Dissolve collagen(x gm) in 0.5 M acetic acid and also
dissolve Hydroxyapatite(x/2 gm) in 0.5 M acetic
acid. And then mix the solutions in equal volumes ,
so ultimately we will have 50% w/v of Collagen:HA
 Then, allow the solution to mix properly or
blending ,until an uniform slurry is formed,
 Allow the mixture to mix on a shaker at 15,000 rpm
at 4°C or it can also be centrifuged at 10,000 rpm
at 4°C so that the temperature does not affect the
triple helical structure of Collagen.
 It is optional to add chemical cross linking
agent(EDAC or any other) at this step at
concentration 5µMol/gm of collagen

15. PREPARATION : FREEZING
PROCESS
 Now pour the slurry into the trays with specific volume
required to fill the trays optimally.
 Then freeze the trays containing the slurry into the
freezer to allow the slurry to freeze and solidify
 Allow the freeze-dryer machine to get stable
temperature of 4°C for 1 hour and then put the tray
containing the mixed slurry into it. Now allow the
trays to get used or come to the usual 4°C temperature
for 1 hour
 Start the freeze-drying process starting from
4°C to-20°C at a constant cooling rate of 1°C/min. This
process would normally take 60 mins cycle. But to
increase the pore size we can alter this cycle to 180 min
so that the cooling rate becomes 0.5°/C

16. PROTOCOL FOR THE SCAFFOLD
PREPARATION: FREEZING
PROCESS
 An introduction of an annealing step, i.e. holding the
temperature at -10°C ,more towards the final freezing
temperature, allows the ice-crystals to grow and then
this ice crystals when sublime create pores of greater
size.
 Then proceed for the drying step , by turning on the
vacuum. When the vacuum pressure reached 200mTorr,
the chamber temperature was raised to 0°C and
maintained for 17 hours
 A secondary drying step followed by increasing the
temperature to 20°C and holding for 30 min
 These conditions induced sublimation of ice crystals in
the slurry, resulting in pore formation in uniform
distribution.

17. Freeze drying process
Cooling rate (1°C/min)
Drying cycle
Final freezing temperature
Annealing step
40 80 120 160
Time (min)

18. STANDARDIZING THE PARAMETERS
OF THE PROTOCOL
 Freezing rate (0.5°C/min to 1°C/min)
 Annealing step (-10°C)
(holding the pre-final freezing temperature)
 Cross linking agent(Concentration of EDAC)
 Amount of Collagen: HA ratio (10%,50%,100%)
 Calculating the porosity in each of the above
parameters

19. PORE ANALYSIS :CHEMISTRY OF THE
PORE SIZE FORMED IN THIS
SCAFFOLD
 To know the exact pore size of the scaffold, we must use a
simple sectioning method.
 Use two identical blade –Hold them about 2 cm apart and
pass it through the scaffold tranversely (Transverse
section )
 Then allow this section to dry completely and then stain
in normal aniline blue stain.
 Wash the excess stain with 70% IPA and then fix the
section on slide with coverslip.
 In this way we can have a rough idea about the pore size
of the formed scaffold.

20.  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

21. EFFICACY OF THE SCAFFOLD
(BIOACTIVITY AND
BIOCOMPATIBILITY)
 To check the efficacy of the scaffold
we must measure the effect of the
scaffold on the growth of mouse
osteoblast cell line MC 3T3 E1 or
human osteoblast cell line
 We can incorporate the semi-solid
scaffold (slurry)on the 6 or 12 -well
plate and allow it to freeze at -20°C,
and then seed the cells on the
scaffold.
 Study the effect of the scaffold on
the growth of the cells and measure
if there is any effect on the growth of
the cells, i.e. stress condition
 Also , if the cells go in a stress
condition the spent medium, in
which the scaffold was seeded with
cells, can be measured for any stress
marker

22. COLLAGEN /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&id=982532
 SyboGraf™- C
Eucare Pharmaceuticals Private Limited
http://www.eucareindia.com/product.html
 Collapat® II
http://www.biomet.fi/ammattilaiset/biomateriaalit/luunkorvikkeet/collapat
 MCH-Cal™ : <850 micron, < 250 micron and < 150 micron
Waitaki biosciences
http://www.waitakibio.com/manufacturer/natural-calcium

23. OTHER COMPETITIVE THREE
DIMENSIONAL POROUS
BIOMATERIALS
 Collagen And 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/prod

24. OTHER PROJECTS
 For Cosmetic application(Iontophoresis)
 For Heavy bleeding –Collagen as a hemostat
 Collagen to be used as a coating material for
tissue culture grade.
 Collagen as a 3-dimensional scaffolds and
sustained drug release or growth factors.

25. FOR HEMOSTAT APPLICATION: HEAVY
BLEEDING
 We can use a inert compound called
kaolin and smectite which is a
mineral component and has an
inherent property of absorbing
water,
 So , this mineral can act as
haemostatic absorbable material for
heavy bleeding.
 QuikClot® TraumaPad™ consists
of a soft, white, double sterile,
three-ply pad impregnated with
kaolin, an inert mineral that does
not contain animal or human
proteins or botanicals.
 QuikClot TraumaPad is indicated
for temporary external use to
control traumatic bleeding and is
also x-ray detectable to ensure
proper removal.

26. FOR COSMETIC APPLICATION
 With the use of Iontophoresis
technology , we use the
transdermal delivery with
the help of specific ionic
strength buffer.
 The delivery of collagen
directly in to the dermis is
possible with this technology

27. AS A TISSUE GRADE COATING
MATRIX.
 Collagen matrix system further serves as the
substrate for the embedding of a single cell
suspension or a small tumor specimen in the
evaluation process of cancer therapy
 Various Tissue culture grade plates are
available which are coated with collagen gels or
films .This can be useful system for growth of
cells efficiently.

28. AS A 3D SCAFFOLD AND
SUSTAINED DRUG RELEASE
 Various growth factors can be crosslinked to the
collagen matrix and serve as a drug delivery
system.
 Even 3-Dimensional scaffolds can be used for
delivery of various growth factors like BMP
(Bone Morphogenetic protein) directly in bones.
 This also serves the purpose of growth of
osteoblast cells and fasten the process of bone
healing

29. FUTURE PLANNING
As soon as we are ready with the
equipments and lab, things can go faster.
Even fast forward >>>>>>

30. CONCLUSION
 Collagen /Hydroxylapatite would serve as good
market product for our company.
 Also, for antimicrobial effect of silver along with
collagen will help to design wound care
management products for diabetic ulcers with
ease.
 Also it would provide a better market profit as
silver is as such an expensive element we are
adding in our product, an added advantage is that
diabetes is widespread in India.
 For ordering materials we have gathered
information and quotation, just need to order them
as soon as possible. chemicals pricing.xlsx
 Have also made a list of requirements for the
starting of the lab. requirement list.xls