1. INTRODUCTION:

Diabetic Mellitus is a metabolic disorder due to its insulin resistance defined as hyperglycemia where type II diabetes mellitus is a major international health concern. Diabetic and its chronic wounds were a threat to public health furthermore diabetes is associated with various chronic complications such as pressure sores, diabetic foot ulcer, and various other wounds. Various treatments for chronic diabetic wounds have been a challenging subject.

Certain existing therapies were effective but still diabetic wounds were associated with the prolonged recovery period. In case of traditional treatment such as creams, gels only the active ingredients will act on the affected area and other dosage forms it only acts as an carrier, however in scaffolds both the active ingredients and carrier will act on the affected area as it’s biodegradable and allow to heal rapidly but in creams, gel it will vanish it off so quickly (1) Hence advancement in tissue engineering have been adopted biologic scaffolds where its uniqueness lies in mimicking the ECM and also gives structural support for the cell attachment, tissue development, repair the damaged tissue (2). Collagen has been used as one of the natural polymers for the effective wound healing collagen molecule is considered as the potential candidate due to its antigenicity, excellent biocompatibility, biodegradability and also possess suitable mechanical and cell binding properties. The collagen molecule, particularly by collagenase enzyme, gets degraded and also its denaturated product at normal physiological temperature by gelatin rapidly gets denaturated. By several nonspecific proteases, these gelatinized fragments get cleaved. The concurrently infiltrating cell will facilitate the new ECM for tissue regeneration. Since collagen is a natural biomaterial it’s an essential component of native ECM possessing excellent control over pore structure, good pore interconnection, high porosity and also have excellent biomechanical properties. Hence based on the statement that noncross linked collagen will spontaneously degrade so it’s considered as one of the requirements in performing the cross-linking in order to maintain the biodegradation rate (3). Polycaprolactone (PCL) can be used as a protective barrier due to its biocompatibility and biodegradability properties it has paid much attention in biomedical applications. It exhibits inimitable mechanical properties due to this, it possesses biocompatible, biodegradable, bioresorbable polymer moreover unique feature in adopting PCL is non toxic and mimicking the fibrous structure of ECM and also absorb wound exudates possess excellent water retention capacity (4) Obesity and hyperlipidemia are one of the factors which affect the wound healing process. Simvastatin is one of the statin derivatives its 3- hydroxy l-3-methylglutaryl coenzyme an (hmg-CoA) reductase inhibitors by reducing the cholesterol levels it improves the tissue repair. The farnesyl pyrophosphate (FPP) synthesis which is an essential element in mevalonate pathway thereby keratinocyte migration is inhibited simvastatin possess essential immunomodulatory effects, anti-inflammatory effects, pleitrophic effects hence it improves the microvascular function,modulates nitric oxide synthase hence it used to treat the infected wounds (5). stain due to their different/various pleiotropic effects such as anti-inflammatory, immunomodulatory, anti-oxidative, anti-bacterial activities improves the microvascular and endothelial function and reperfusion. Various research studies have been demonstrated simvastatin in the diverse phase of the healing process. Simvastatin improves the VEGF production and secretion hence it has beneficial effect increased wound breaking strength and elevated no live in the wound healing process (6). In this work we have fabricated simvastatin loaded in polycaprolactone-collagen scaffolds via freeze-drying method to enhance the cell attachment, vascularisation, tissuerepair during wound healing for the treatment of diabetic foot ulcer. Morphology, Stability, degradation, cell viability, cell attachment and in vivo wound healing efficacy of these simvastatin loaded PCL-collagen scaffolds have been characterized and compared with controls.

2. MATERIALS AND METHODS:

2.1. Materials:

Simvastatin was purchased from SD Fine Chemicals, Mumbai, India, Collagen, Polycaprolactone were procured from SD Fine Chemicals, Mumbai, Glacial acetic acid, Potassium dihydrogen ortho phosphate, Sodium hydroxide pellets, Ethanol was purchased from SD Fine Chemicals, Mumbai, India.

2.1.1. Equipment:

Lyophilizer purchased from Sub-zero lab instruments, Chennai

2.2. Methods:

2.2.1.Fabrication of the composite scaffold:

1g of collagen was added to 100ml of water and left for 3days for mass formation. (Solution-1), then to the 1gm of simvastatin 100ml of chloroform and 2gm of polycaprolactone was added in favorable conditions which are stirred at 500rpm(solution-2). Solution 1(50ml) and solution2(50ml) were mixed with continuous stirring. The product obtained was kept for a deep freeze for 3 days. Lyophilization process was carried out for 1 week of duration. The scaffolds obtained as the final product was kept in a desiccator for storage purpose.

2.2.2. Characterization of the composite scaffold:

2.2.2.1. Scanning Electron Microscopy:

Field emission scanning electron microscopy (FE-SEM, Hitachi S-4800) used for determination of cross-section morphologies of porous scaffolds. Primarily, the samples (5 × 5 mm) of scaffolds were coated with a thin layer of gold then placed on the adhesive stub. 10 kV is an operational voltage for FE-SEM (14). The pore size is determined by using SEM images (3). In this study, fifty pores are selected randomly and pore size is calculated. The average diameter of fifty randomly selected pores was resolute. From each group of scaffolds, three scaffolds were selected of three different cross-sections and pore size was estimated (7).

2.2.2.2. Differential scanning calorimetry (DSC):

The thermal stability of simvastatin containing polycaprolactone-collagen scaffolds was assessed using differential scanning Calorimetry (DSE-50, Shimadzu, Japan) under a nitrogen atmosphere at a heating rate of 10^ᵒC/minute to examine their thermodynamic characteristics. The sample is taken in the metallic pan and it has to be loaded within the 0.2-0.8mg and should be sealed properly(3).

There are two compartments in DSC one is reference compartment and the other is a sample compartment. The reference compartment should be maintained blank and sample compartment will be loaded with the sample. The instrument was run till it reaches 300⁰C under the nitrogen environment. The thermograms which are obtained are considered for analysis (15).

2.2.2.3. Porosity:

The porosity of the scaffolds can be determined by liquid displacement method. In this method, ethanol was used as displacement liquid as it can effortlessly penetrate into the pores of scaffolds, did not stimulate contraction and swelling of scaffolds(8).Simvastatin containing scaffolds with characteristic polycaprolactone-collagen ratios before and after the cross-linking treatment is subjected for determination of porosity by using method followed by Shimadzu et. al. Ethanol displacement method is used to determine the pore volume (Vp) and geometric volume (SV) of the scaffolds disc is measured by determining the diameter and heights. Air bubbles are removed by immersing the weighed scaffolds (Ww) in absolute ethanol at room temperature and then kept in desiccators under reduced pressure for five minutes (16). Samples were taken out in a subsequent manner, then wiped with filter paper to remove excess of ethanol and weighed instantly (Wd). The porosity of scaffolds is calculated according to the following equation.

Ww - Wd

Porosity (%) = ^{-----------------------------------------------------------------------------------------} X100

Where, SV is the scaffold volume; W_w and W_d are the wet and initial dry weights of the scaffolds, respectively.

2.2.2.4. Water absorption test:

The scaffolds were weighed as dry weight (𝑊o). The scaffold sample then incubated in phosphate-buffered saline (PBS) at pH 7.4 at 37⁰C for 2h (18). The swollen scaffold then weighed after removing the excess water by blotting with filter paper. Then proceed for lyophilization and reweighing of scaffold sample. The water content of scaffold sample calculated using the following formula(9).

[We - Wo]

Ead = ^{--------------------------------------------------------------------------------------------------------} X100

Where𝐸ad is the percentage water adsorption of polycaprolactone-collagen at equilibrium. 𝑊𝑒 and 𝑊o represent the weight of the polycaprolactone-collagen scaffold at equilibrium and the initial weight of the polycaprolactone-collagen scaffold, respectively (3).

2.2.2.5. Matrix degradation:

To measure the biodegradability, the initial weight of scaffold was measured and was immersed in a phosphate buffer solution (17). Then the scaffold was kept at 37⁰C in the incubator and the measurements were taken third and sixth day by determining the final weight. Finally, the percentage loss was determined using all the above data(10).

W₀ -W_t

Degradation % =--------------------------- X 100

W₀

2.2.2.6. In vitro drug release studies and kinetics:

The composite scaffolds (3 × 3cm) were immersed in 20 mL phosphate buffer solution having pH 7.4 at 37^∘C (19). the supernatant was taken out periodically and replaced by an equivalent volume of BPS. The drug release is determined by UV-visible spectrometer at 420nm by using simvastatin standard curve in ethanol. The percentage of Simvastatin released was determined(3).

2.2.2.7. In vitro cytotoxicity:

The cytotoxicity studies of the composite scaffold are carried out on mouse fibroblast cells by indirect MTT method (20). The cells are embedded into 96-well microplates at a density of 103 cells/well at 37⁰C under a humidified atmosphere containing 5% CO₂. Triplicates of the sterilized sample are taken and further incubated in serum-containing media for 24h at 37^∘C. from each sample 100 𝜇L of the media is taken, followed by relocating it into each well then incubated for 24h. Then MTT solution is added to each well plate and further incubated for 4 h at 37^∘C. afterwards, the medium was removed and violet crystals precipitated will be solubilized with DMSO. After shaking the solution slowly for 5min, the absorbance of each well will be observed of each well plate using Eliza reader at 570nm. The viability of ctheell will be determined as a percentage of the control (11).

2.2.2.8. Cell proliferation assay:

Fibroblast 3T3 cells were seeded in a 96-well culture plate in a humidified 5% CO₂atmosphere (21). Cells were serum starved for 24h and then incubated with 5, 10, 25 and 50μg/ml of Test formulation and further incubated for 24h. After 24h 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added and formazan product was dissolved in 100μl of DMSO. Absorbance at 570 nm was measured with a microtiter plate reader and cell viability was determined by trypan blue exclusion method(12) (13).

2.2.2.9. Cytotoxicity assay:

The ability of the cells to survive a toxic insult has been the basis of most cytotoxicity assays. Determining the mitochondrial synthesis by MTT assay. This assay is based on the assumption that dead cells or their products do not reduce tetrazolium (22). The assay depends both on the number of cells present and on the mitochondrial activity per cell. The cleavage of MTT to a blue Formosan derivative by living cells is clearly a very effective principle on which the assay is based. The principle involved is the cleavage of tetrazolium salt MTT (3-(4,5 dimethyl thiazole-2 yl)- 2,5-diphenyl tetrazolium bromide) into a blue colored product (formazan) by mitochondrial enzyme succinate dehydrogenase. The numbers of cells were found to be proportional to the extent of formazan production by the cells used (Francis and Rita, 1986).

Procedure:

The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0x105 cells/ml using DMEM medium containing 10% FBS. To each well of a 96 well microtitre plate, 100µl of the diluted cell suspension (approximately 10,000 cells/well) was added. After 24h, when a partial monolayer was formed, the supernatant was flicked off (23), the monolayer was washed once with medium and 100ml of different test sample concentrations prepared in maintenance media were added per well to the partial In vitro cytotoxicity studies TC Lab., Dept. of Pharm. Biotechnology monolayer in microtitre plates. The plates were then incubated at 37⁰C for 48 h in 5% CO₂ atmosphere, and microscopic examination was carried out and observations recorded every 24h.After 48 h,(24) the sample solutions in the wells were discarded and 20 ml of MTT (2mg/ml) in MEM-PR (MEM without phenol red) was added to each well. The plates were gently shaken and incubated for 3h at 37⁰C in 5% CO₂ atmosphere. The supernatant was removed and 50ml of iso-propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540nm. The percentage growth inhibition was calculated using the following formula and concentration of drug or test samples needed to inhibit cell growth by 50% values were generated from the dose-response curves for each cell line(13).

% cell viability test can be calculated by

Mean O Do find individual test group

^{------------------------------------------------------------------------------------------------------------------------------------------}×100

Mean O D of control group

3. RESULTS AND DISCUSSION:

3.1. Formulation of scaffolds:

Simvastatin–chloroform solution and polycaprolactone- chloroform solution is prepared. Either of the solutions should be added to other by continuous stirring using magnetic stirrer at cold temperature. The above solution was added to a freshly prepared collagen solution and was ultimately mixed in Remi stirrer. The final solution after mixing will be proceeding for lyophilization.

3.2. Characterization of the composite scaffolds :

3.2.1 Scanning Electron Microscopy:

Figure-1 SEM samples for scaffold

The SEM analysis revealed interconnected pores of different size and flat, relatively smooth walls. The SEM images as shown in the figure shows the average diameter of the pores on the surface of scaffolds were 370 micrometers. The SEM images of simvastatin loaded polycaprolactone - collagen scaffold shows the appearance of incorporation of simvastatin in the scaffold, confirmed the homogenous distribution of simvastatin throughout the scaffold as shown in figure 1.

3.2.2 Porosity studies:

Porosity is the percentage of void space in the scaffolds. The porosity of the scaffold will assist cell migration and vascularization which is essential for tissue formation. It also controls the mass transfer of nutrients and metabolic waste products to the cell. Porosity is characterized by the presence of the open pores which relates to the permeability and surface area of the porous structure. High porosity will provide a favorable biological environment to the wound healing. The porosity of simvastatin loaded in polycaprolactone-collagen scaffolds was found to be 90.4% as shown in table 1.

Table 1: Porosity studies

S. No.	Type of scaffold	% porosity
1	Non cross-linked (1%)	90.4

3.2.3 Water absorption test:

The water absorption property of material influences not only the maintaining of collagen scaffolds shape and form but also affects the cell growth. If high water absorption materials were used, the scaffolds might saturate with water and expand causing deform and affecting the proliferation and division of cells. The concentration of collagen increase will decrease water absorption. The decrease of pore size may cause capillary phenomenon and increase water absorption. The percentage water absorption found to be more in simvastatin loaded in polycaprolactone- collagen scaffold as shown in figure 9.

Table 2: Water absorption test

S. No.	Type of scaffold	% water absorption
1	Noncross-linked S1 (1%)	50.95

3.2.4 Matrix degradation studies:

To measure the biodegradability, the initial weight of scaffold was measured and was immersed in a phosphate buffer solution. Then the scaffold was kept at 37⁰C in the incubator and the measurements were taken third and sixth day by determining the final weight. Finally, the percentage loss was determined using all belowdata as shown in table 10.

3.2.5 In vitro drug release studies and kinetics:

Drug release profiles of the simvastatin loaded polycaprolactone- collagen scaffold was studied over a period of 168h with aliquots taken from the tubes at 60min intervals initially for the first 3h, followed by every 3h, 6h and then 12h interval thereafter. From the data in Fig, there was a slower release in all formulation followed by a sustained release and then a plateau. The cross-Linked Scaffold has a significant slower release in comparison to the non-crosslinked scaffold, this is because of cross-linking of the scaffolds the chemical and mechanical bonding is high compared to noncrosslinked scaffold. The release rate of simvastatin in PBS is determined. The percentage release of simvastatin was found to be as shown in table 11 and represented in figure 7:

Table 4: In vitro drug release studies and kinetics

S. No	Time (mins)	The release of the non-crosslinked scaffold
1	1	4.74
2	2	14.27
3	3	19.42
4	4	24.5
5	5	29.54
6	6	34.84
7	7	40.32
8	12	46.8
9	24	51.80
10	48	55.9
11	72	65.22
12	96	71.38
13	120	77.72
14	144	84.44
15	168	91.12

Figure No 2:in vitro release of the non-crosslinked scaffold

3.2.6. In-Vitro Cytotoxicity Assay:

The percentage growth inhibition was calculated using the following formula and concentration of drug or test samples needed to inhibit cell growth by 50% of values were generated from the dose-response curve for each cell line.

% cell viability test can be calculated by

Mean O Do find individual test group

^{------------------------------------------------------------------------------------------------------------------------}×100

Mean O Do f control group

Table 5: In-vitro cytotoxicity assay

S. No.	Sample Description	IC₅₀µg/ml
1.	Drug	164.38
2.	Placebo	279.92
3.	Non-Crosslinked Scaffold	289.34

Figure No 3: Determination of cytotoxicity by MTT assay for NCS

Figure No 4: Determination of cytotoxicity by MTT assay for placebo

Figure no 5: Determination of Cytotoxicity by MTT assay for drug

3.2.7. Cell Proliferation Assay:

Table-6: Table No.-a (After 1h)

S. No	Sample	Time in hours	Percentage cell growth
1	Control	1	100
2	Placebo scaffold	1	100
3	Drug-loaded scaffold	1	100

Table-b (After24h)

S. No	Sample	Time in hours	Percentagecell growth
1	Control	24	100
2	Placebo scaffold	24	100
3	Drug-loaded Scaffold	24	102

Table-c (After 48h)

S. No	Sample	Time in hours	Percentage cell growth
1	Control	48	100
2	Placebo scaffold	48	103
3	Scaffold	48	111

Table No.-d (After72h)

S. No	Sample	Time in hours	Percentage cell growth
1	Control	72	100
2	Placebo scaffold	72	110
3	Scaffold	72	128

· The cell viability test was performed by using 3T3-L1 cells because these are the cells which can be differentiated to form keratinocytes and forms the extra cellular matrix. We can quantify cell growth. The above table indicates the cell growth in percentages,

· At the end of the 1^st day i.e. after 24 h, cell growth is not higher because of time taken by the cells to adopt in new habitat which can be termed as a lag phase.

· At the end of 2^nd day i.e. after 48 h, rapidity in the cell growth was initiated because cells have started absorbing the nutrients, which can be described as the initial portion of the logarithmic phase.

· At the end of 3^rd day i.e. after 72 h, the cell growth was found to be greater when we compared with placebo and control due to the presence of the drug, which explains that the cells are in logarithmic phase.

· The scaffold base contains collagen and polycaprolactone, which are protein and carbohydrate respectively. These will give support by supplying the nutrients to the growing cell.

Figure 6: Cell proliferation assay

3.2.8. Differential Scanning Calorimetry (DSC):

Collagen has shown the two peaks one is at the 110⁰C and another peak at 160⁰C

Polycaprolactone at 65⁰C, Simvastatin at 140⁰C. The physical mixture which contains the simvastatin and polycaprolactone at 140⁰C and 65⁰C respectively.

The formulation contains all the peaks at 110⁰C and 160⁰C which are for collagen, peak at 140⁰C which resembles the simvastatin and peak at 65⁰C. This shows that there is no change in the peaks by that we can confirm that all the excipients are compatible with each other.

Figure 7: Thermograms for drug, excipients, and formulation

4. CONCLUSION:

This work deals with the investigation carried out on the Design and Development of simvastatin-Loaded in polycaprolactone- collagen scaffold for the treatment of Diabetic foot ulcer, to decrease dosing frequency, maintain prolonged therapeutic levels of the drug, synergistic and faster-wound healing effects. Collagen scaffolds were prepared by the freeze-drying method and the effect of certain process and formulation variables such as polymer concentration, solvent concentration, solvent purity, stirring conditions and time were considered.

Collagen non-cross-linkedscaffoldswere evaluated for SEM studies, porosity, water absorption, matrix degradation and in vitro release studies. The in vitro data was transformed and interpreted at graphical interface In order to elucidate mode and mechanism of drug release. Compatibility of the selected polymer and simvastatin were carried by DSC peak matching method. Non-crosslinked, at the end of the 7^th day we found 85.34% drug was released. By this, it can be concluded that non-cross-linked scaffolds show the prolonged release. The non-cross linked scaffold has shown the better IC50 values when compared to drug and placebo and the value was found to be 289.34.Non-cross-linked scaffold has shown an increase in cell growth by 28% from its initial value i.e 100% at the end of 3^rd day.

All the above investigations brought out many facts which lead to following conclusions. The prepared simvastatin-polycaprolactone Collagen scaffolds satisfied the properties which are required the ideal wound dressing in terms of porosity, water absorption, bio degradation, drug release, cytotoxicity, and cell proliferation. Which can justify the tissue regeneration? Hence the present study states that simvastatin is having all the properties regarding to treat the Diabetic Foot Ulcer without producing the resistance.

5. DISCLOSURE OF INTEREST:

The author declaresthat thereis no conflict of interest involved in this research work. The authors alone are responsible for the content and writing of the paper.

6. REFERENCE:

1. Karri VVSR, Kuppusamy G, Talluri SV, Yamjala K, Mannemala SS, Malayandi R. Current and emerging therapies in the management of diabetic foot ulcers. Curr Med Res Opin [Internet]. 2016;32(3):519–42. Available from: http://www.tandfonline.com/doi/full/10.1185/03007995.2015.1128888

2. Landén NX, Li D, Ståhle M. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci. 2016;73(20):3861–85.

3. Mahmoud AA, Salama AH. Norfloxacin-loaded collagen/chitosan scaffolds for skin reconstruction: Preparation, evaluation and in-vivo wound healing assessment. Eur J Pharm Sci [Internet]. 2016; 83:155–65. Available from: http://dx.doi.org/10.1016/j.ejps.2015.12.026

4. Mir M, Najabat M, Afifa A, Ayesha B, Munam G, Shizza A. Synthetic polymeric biomaterials for wound healing: a review. Prog Biomater [Internet]. 2018; (Mostow 1994). Available from: https://doi.org/10.1007/s40204-018-0083-4

5. Cínthia A, Irami R, Filho A, Damasceno BPGL, Sócrates E, Egito T. Simvastatin improves the healing of infected skin wounds of rats 1 A Simvastatina melhora a cicatrização de feridas infectadas da pele de ratos. 2007;22(Supplement 1):57–63.

6. Farsaei S, Khalili H, Farboud ES. Potential role of statins on wound healing: review of the literature. 2012;238–47.

7. Baxter R, Hastings N, Law a., Glass EJ. [ No Title]. Anim Genet. 2008;39(5):561–3.

8. Thangavel P, Ramachandran B, Muthuvijayan V. Fabrication of chitosan / gallic acid 3D microporous scaffold for tissue engineering applications. 2015;750–60.

9. Sultana N, Khan TH. Water absorption and diffusion characteristics of nanohydroxyapatite (nHA) and poly(hydroxybutyrate-co-hydroxyvalerate-) based composite tissue engineering scaffolds and nonporous thin films. J Nanomater. 2013;2013.

10. Kumar PTS, Srinivasan S, Lakshmanan V, Tamura H, Nair S V, Jayakumar R. ␤ -Chitin hydrogel / nano hydroxyapatite composite scaffolds for tissue engineering applications. Carbohydr Polym [Internet]. 2011;85(3):584–91. Available from: http://dx.doi.org/10.1016/j. carbpol.2011.03.018

11. Sun X, Wang J, Wang Y, Zhang Q. Collagen-based porous scaffolds containing PLGA microspheres for controlled kartogenin release in cartilage tissue engineering. Artif Cells, Nanomedicine, Biotechnol [Internet]. 2017;0(0):1–10. Available from: https://doi.org/10.1080/21691401.2017.1397000

12. Vernon RB, Gooden MD, Lara SL, Wight TN. Microgrooved fibrillar collagen membranes as scaffolds for cell support and alignment. 2005; 26:3131–40.

13. Demirci S, Doğan A, Demirci Y, Şahin F. In vitro wound healing activity of methanol extract of Verbascum speciosum. 2014;7(3):37–44.

14. Saurabh Mehta. Review of Heterocyclic Scaffolds for the Inhibitors of ATP synthase. Asian J. Research Chem. 2018; 11(2):505-508.

15. Radhika G, Sreelakshmi Divya P, Prashanth Reddy G, Venkatesh P, Ravindra Reddy K. An Overview on Regenerative Medicine. Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 727-728.

16. Hisham A. Abbas. Diabetic Foot Infection. Research J. Pharm. and Tech. 8(5): May, 2015; Page 575-579.

17. P. Maheshwari, D. Pavithra, Neethu. T. T, T.S. Shanmugarajan, P. Shanmugasundaram. Study on Health Outcomes in Diabetic Patients - Association Between Diabetic Foot Ulcer and Psychological Distress. Research J. Pharm. and Tech. 2017; 10(1): 44-48.

18. Radhika Chelamalla, Ajitha Makula. Molecular docking studies and ADMET Predictions of Pyrimidine Coumarin Scaffolds as Potential IDO Inhibitors. Asian J. Research Chem. 2017; 10(3):331-340.

19. Bonshikachatterjee, Nivetha. A, Mohanasrinivasan. V. Immobilization of β-galactosidase in Chitosan-Alginate composite scaffolds and optimization of lactose hydrolysis. Research J. Pharm. and Tech 2018; 11(4): 1480-1485.

20. Smriti Agarwal, Vinayak Jhunjhunwala, G. Priya. Fabrication and Morphological Analysis of Gelatin-Alginate Scaffolds. Research J. Pharm. and Tech 2018; 11(9): 3816-3818.

21. Saumya S, Agila Anbuselvan, Poorva S, G. Priya. A Review on 3D Printing Techniques and Scaffolds for Auricular Cartilage Reconstruction. Research J. Pharm. and Tech 2018; 11(9): 4179-4186

22. Keerthic Aswin S, Jothishwar S, Visvavela Chellaih Nayagam P, G. Priya. Scaffolds for Biomolecule Delivery and Controlled Release–A Review. Research J. Pharm. and Tech 2018; 11(10): 4719-4730.

23. Hisham A. Abbas, Mona A. El-Sayed, Laila M. Al-Kadi, Amany I. Gad. Diabetic foot infections in Zagazig University Hospital: bacterial etiology, antimicrobial resistance and biofilm formation. Research J. Pharm. and Tech. 7(7): July 2014 Page 783-788

24. P. Maheshwari, D. Pavithra, Neethu. T. T, T.S. Shanmugarajan, P. Shanmugasundaram. Study on Health Outcomes in Diabetic Patients - Association Between Diabetic Foot Ulcer and Psychological Distress. Research J. Pharm. and Tech. 2017; 10(1): 44-48.

25. Robson MC, Mustoe TA, Hunt TK. The future recombinant growth factors in wound healing. Am J Surg 1998;2(suppl 1):80-2S

Received on 21.12.2018 Modified on 01.02.2019

Research J. Pharm. and Tech. 2019; 12(6): 2637-2644.

DOI: 10.5958/0974-360X.2019.00441.4

Matrix degradation study for non-crosslinked scaffold
S.no	Scaffold	Day	Initial weight	Final weight	Percentage loss
1	Non-crosslinked	3	400	265	33.75
2	Non-crosslinked	6	400	162	59.5