CURRENT PROJECTS

Presented By: Akshita Vyas

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.

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)

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

Silver nano powder Nanoshel LLC - Intelligent Materials Pvt Ltd

COMPETITIVE PRODUCTS (COLLAGEN & SILVER NANO)

Smith and Nephew Colactive Ag Collagen Dressing 

Puracol Plus AG+ Wound Dressing with Antibacterial Silver

SilvaKollagen Gel Silver Antimicrobial Collagen

SORE-TREAT-SLR –Helix pharma(Collagen Based Cream of  Silversulfadiazine)

Eucarepharmaceuticals Collagen Film With Silver SulphadiazineNeuskin-FS

.

NEW INSIGHT IN TO BONE DEFECTS

Collagen Hydroxyapatite scaffold

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

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

WHY COLLAGEN AND HYDROXYAPATITE ?

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.

COLLAGEN /HA COMPOSITE

(Left) Fiberized hydroxyapatite and collagen compound composite immediately after adjustment

(Right) Hydroxyapatite and collagen compound composite made porous

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

Collagen /Hydroxyapatite scaffolds Work going on . . . . .

HYDROXYLAPATITE Chemical formula Ca5(PO4)3OH CAS No 12167-74-7 SYNONYMS

1. PENTA-CALCIUM HYDROXIDE TRIPHOSPHATE

2. TERT-CALCIUM PHOSPHATE3. TRI-CALCIUM DI-ORTHOPHOSPHATE4. TRI-CALCIUM (ORTHO)PHOSPHATE5. TRI-CALCIUM PHOSPHATE6. TRI-CALCIUM PHOSPHATE (BETA-FORM)7. CELLULOSE HYDROXYAPATITE8. CALCIUM HYDROXIDE PHOSPHATE9. CALCIUM PHOSPHATE TRIBASIC10. CALCIUM ORTHOPHOSPHATE;11. BETA-CALCIUM PHOSPHATE TRIBASIC12. BETA-TRICALCIUM PHOSPHATE

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

PROTOCOL FOR THE SCAFFOLD 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

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.

Freeze drying process

40 80 120 160Time (min)

Cooling rate (1°C/min)

Final freezing temperature

Drying cycle

Annealing step

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

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.

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

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

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

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/product/fibracol-plus-collagen-wound-dressing-with-alginate_5049.html

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.

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.

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

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.

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

FUTURE PLANNING

As soon as we are ready with the equipments and lab, things can go faster.

Even fast forward >>>>>>

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