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Collagen application

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  • Good work Ms.Vyas !

    Nilesh Gandhi

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  1. 1. CURRENTPROJECTSPresented By: Akshita Vyas
  2. 2. SCENARIO OF WOUND CARE MANAGEMENT ININDIA 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 Indias 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. 3. FOR DIABETIC FOOT ULCERS WE REQUIRE AMORE ADVANCED WOUND CARE PRODUCTTHE ANSWER TO IT IS SILVER NANO PARTICLES It is proven fact that AgNP haveproperties like good conductivity,chemical stability, catalytic , antibacterialactivity, antifungal, anti-viral,antiinflammatory,so they have beenused in various medical devices. (Mukherjee et al., 2001; Sondi andBranka, 2004; Chen and Schluesener,2008) It has been found that smallerparticles generally present an enhancedantibacterial activity if compared withtheir larger counterparts. The range of the size other medicaldevices uses is 10-100 nm(20,40,60,80,100nm)
  4. 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. 5.  Silver nano powder Nanoshel LLC - Intelligent Materials Pvt Ltd
  6. 6. COMPETITIVE PRODUCTS Puracol Plus AG+ Wound (COLLAGEN & SILVER Dressing with NANO) Antibacterial Silver Smith and Nephew Colactive Ag Collagen Dressing  Eucarepharmaceuticals Collagen Film WithSORE-TREAT-SLR – Silver Sulphadiazine SilvaKollagen GelHelix pharma Neuskin-FS Silver Antimicrobial(Collagen Based Cream Collagenof  Silversulfadiazine) .
  7. 7. NEW INSIGHT IN TOBONE DEFECTSCollagen Hydroxyapatite scaffold
  8. 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
  9. 9. WHY COLLAGEN AND HYDROXYAPATITE ?The natural bones contain mainlycollagen and Hydroxyapatite. Thatis the reason because manyresearchers try for(nano)Hydroxyapatite/collagencomposite for hard tissue repairingComposition of Bone Tissue 25% Water 25% Protein or organic matrix 95% Collagen Fibers 5% Chondroitin Sulfate 50% Crystallized Mineral Salts Hydroxyapatite
  10. 10. ADVANTAGES OF COLLAGEN /HACOMPOSITES 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. 11. COLLAGEN /HA COMPOSITE (Left) Fiberized hydroxyapatite and collagen compound composite immediately after adjustment (Right) Hydroxyapatite and collagen compound composite made porous
  12. 12. Collagen /Hydroxyapatite scaffolds Work going on . . . . . Collagen/Hydroxylapatite Scaffolds protocol Standardizing the protocol on small scale Studying the Detailed Chemistry of the pore sizeformed in this scaffold Studying the efficacy of the formed scaffold
  14. 14. PROTOCOL FOR THE SCAFFOLDPREPARATION: SLURRYPREPARATION 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. 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. 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. 17. Freeze drying processCooling rate (1°C/min) Drying cycle Final freezing temperature Annealing step 40 80 120 160 Time (min)
  18. 18. STANDARDIZING THE PARAMETERSOF 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. 19. PORE ANALYSIS :CHEMISTRY OF THEPORE SIZE FORMED IN THISSCAFFOLD 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. 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. 21. EFFICACY OF THE SCAFFOLD(BIOACTIVITY ANDBIOCOMPATIBILITY) 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. 22. COLLAGEN /HA PRODUCTS INMARKETProduct/ Company Name HydroxyColl Ossfill SEWON CELLONTECH CO., LTD. SyboGraf™- C Eucare Pharmaceuticals Private Limited Collapat® II MCH-Cal™ : <850 micron, < 250 micron and < 150 micron Waitaki biosciences
  23. 23. OTHER COMPETITIVE THREEDIMENSIONAL POROUSBIOMATERIALS 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. 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
  24. 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. 25. FOR HEMOSTAT APPLICATION: HEAVYBLEEDING 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. 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. 27. AS A TISSUE GRADE COATINGMATRIX. 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. 28. AS A 3D SCAFFOLD ANDSUSTAINED 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. 29. FUTURE PLANNINGAs soon as we are ready with theequipments and lab, things can go faster.Even fast forward >>>>>>
  30. 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