PraseodymiumâCobaltite-Reinforced Collagen as Biomimetic

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Praseodymium-cobaltite Re-inforced Collagen as Biomimetic Scaffolds for Angiogenesis and Stem cell differentiation for Cutaneous Wound healing Vinu Vijayan, Sreelekshmi Sreekumar, Fathe Singh, Dharunya Govindarajan, Rachita Lakra, Purna Sai Korrapati, and Manikantan Syamala Kiran ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00405 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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ACS Applied Bio Materials

Praseodymium-cobaltite Re-inforced Collagen as Biomimetic Scaffolds for Angiogenesis and Stem cell differentiation for Cutaneous Wound healing Vinu Vijayanǂ$, Sreelekshmi Sreekumarǂᶲ, Fathe Singhǂᶲ, Dharunya Govindarajanǂ, Rachita Lakraǂ, Purna Sai Korrapatiǂᶲ and Manikantan Syamala Kiranǂᶲ*$ ǂ Biological

Materials Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai-

600020, Tamil Nadu, India. $

University of Madras, Chennai, Tamilnadu, India

ᶲAcademy

of Scientific and Innovative Research, CSIR-Central Leather Research Institute,

Adyar, Chennai -600020, Tamil Nadu, India * Corresponding Author- [email protected], [email protected] Keywords- Collagen, Praseodymium, Cobalt, Angiogenesis, Stem cell differentiation, Wound healing, Biomaterial, Tissue engineering

Abstract

The present study describes the fabrication of collagen re-inforced with praseodymium-cobaltite nanoparticles for wound healing applications. Praseodymium-cobaltite nanoparticles (PCNP) ACS Paragon Plus Environment

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reinforced with collagen resulted in increased thermal stability and decreased proteolytic susceptibility to collagen. Circular dichroism spectroscopy and ATR-FTIR (Attenuated Total Reflection Fourier Transform Infrared) spectroscopy analyses confirms the intact structural integrity of the collagen sheets after cross-linking with praseodymium-cobaltite nanoparticles. Cross-linked collagen has shown to possess biocompatibility, less protein adsorption behavior and hemo-compatibility which are the desirable properties of a wound dressing material. The nanoparticle cross-linked collagen sheets provided proper matrix elasticity that promotes mesenchymal stem cell attachment and angiogenesis. Further, the scaffold promoted tube formation in endothelial cells. The enhancement of angiogenesis is considered to be brought about by the therapeutic potential of nanoparticle formulation. Praseodymium-cobaltite nanoparticles crosslinking increased the ductility of collagen sheets for pro-angiogenic and stem cell differentiation ability. Also, the Praseodymium-cobaltite cross-linked collagen sheets have shown to induce mild level of ROS (Reactive Oxygen Species) generation in DCFH-DA(2’, 7’Dichlorodihydrofluorescein diacetate) assay which is beneficial for angiogenesis as well as wound healing. This study paves the way for exploring the therapeutic potential of rare-earth based nanoparticles for tissue engineering applications as an alternative for traditional wound healing materials.

Introduction Biomaterials for soft tissue engineering are made to assist the recovery of physiological homeostasis in host tissue after tissue damage. Several kinds of natural as well as synthetic polymers are used for tissue engineering applications1. Collagen, an ECM protein present in mammals is the preferred natural polymer for the fabrication of such biomaterials due to several merits such as high biocompatibility, lower immunogenecity and less chances of rejection. Still,

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its use in native form is a challenge due low mechanical stability, thermal stability and high susceptibility towards proteolytic enzymes2. Cross-linking is the widely used approach to strengthen the native collagen making it suitable for biomaterial applications3. Chemical and natural cross-linking agents such as glutaraldehyde4, formaldehyde4, 1-Ethyl-3-(3-dimethyl amino propyl) carbodiimide (EDC) 5etc are being used for crosslinking of collagen. Chemical assisted cross-linked collagen offers high mechanical stability and less proteolytic susceptibility4 but is compromised with regards to its biocompatibility4. Leaching out of cross-linking agents from the collagen biomaterial causes complications in the host tissue5. Toxicity and calcification arising from such percolations can cause impaired immune response6,7. Thus, investigation into an alternative cross-linking agent, of chemical or natural origin with high biocompatibility, highmechanical and thermal strength and less susceptibility towards protease enzymes is of great importance. Metal nanoparticles are gaining importance as cross-linkers of collagen for biomedical applications8. They have been reported to form multiple cross-linking sites for inducing collagen fibrillation8. Metal nanoparticles have been shown to impart good biomechanical and thermal stability to collagen scaffolds and use of metal nanoparticles as cross-linkers has shown to improve the therapeutic efficacy of collagen scaffolds9,10. The therapeutic property of metal nanoparticles is expressed in the biomaterial scaffolds where it not only acts as cross-linkers but also functions as bioactives10. However, side effects of these metal nanoparticles have been reported once the biomaterial gets resorbed into the host tissue 11. In this scenario, rare-earth elements are superior owing to their highly biocompatible nature12. The effect of rare earth nanoparticles on collagen cross-linking is less explored. The high thermal stability and non-ROS production of the rare-earth metal nanoparticles are other soothing properties for their use in biological applications13. The higher biocompatibility of these ACS Paragon Plus Environment

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nanoparticles may cause differentiation of stem cells into endothelial cells giving a faster wound healing effect. In this study, a bimetallic nanoparticle system with Praseodymium nanoparticle from the lanthanide group as the core of the bimetallic nanoparticle system and cobalt as additive was used for cross-linking of collagen. A bimetallic nanoparticle system can give therapeutic effects from both the participating metal nanoparticles rather than from a single nanoparticle with limited beneficial effect. Several of the biological benefits of Praseodymium are reported in literature. The effect of praseodymium on alteration of surface permeability in prokaryotic oraganisms14and antiinflammatory effect of its inorganic salts15 are reported. Praseodymium along with other lanthanides such as erbium has shown to induce anticancer effect when used as complex with Schiff’s base16. Alloys of Praseodymium with magnesium have shown to be biocompatible and bio-absorbable in implant studies performed on rabbits17.Corrossion resistant Pr-TiN coatings are reported to exhibit positive results on endothelial cell proliferation and blood compatibility18. Other biological properties of Praseodymium including its wound healing potential are less explored. Cobalt even though a heavy metal, has shown to have pro-angiogenic effect19 and is serving the body as trace element and cofactors of several enzymes. It has shown to possess antibacterial and anti-cancer activities in bimetallic form as well as in the form of complexes20,21. But, the biological effects of combination of Praseodymium and cobalt are not yet studied. The bimetallic Praseodymium-cobaltite nanoparticles positively showed higher biocompatibility with angiogenic effect. The nanoparticle system caused cross-linking of native collagen and the sustained release of praseodymium and cobalt oxide nanoparticles from such collagen sheets facilitated angiogenic effects and stem cell differentiation properties while compared to the control native collagen and glutaraldehyde-reinforced collagen. The mechanical stability and susceptibility towards proteolytic enzymes was observed to be superior in PraseodymiumACS Paragon Plus Environment

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cobaltite nanoparticle reinforced collagen sheet. A faster healing against soft tissue damage was observed in invivo wound healing assay. Experimental Materials and methods All the chemicals used were procured from Sigma Aldrich, USA and HiMedia Labs, India unless otherwise mentioned. Endothelial cell line, EA.hy926 was purchased from ATCC, United States. All tissue culture wares were procured form TPP, Switzerland. Synthesis of Praseodymium-cobaltite nanoparticles To 0.1M of Pr (NO3)3.6H2O, 0.07M of Co (NO3)2. 6H2O was added and the solution was reduced with 0.04M citric acid by stirring at 900C overnight22. The gel formed was calcinated at 8000C and centrifuge washed with acetone, ethanol and distilled water at 7000rpm for 10 minutes each. The washed naoparticles was dried in oven at 1000C and crushed with mortar and pestle yielding fine powder of praseodymium-cobaltite nanoparticles. Characterization of Nanoparticles Particle Size analysis The particle size of the Praseodymium-cobaltite nanoparticles was measured with a Dynamic Light Scattering instrument, Zetasizer v.7.11 of Malvern Instruments, UK. The nanoparticles were made into a colloidal dispersion in aqueous medium and the intensity of scattered incident light was detected. A measuring temperature of 250C with duration of 10s was set. Powder X-Ray Diffraction Rigaku mini flex-II desktop diffractometer with Cu κα 1.54Å radiation was used for measuring the X-Ray Diffraction pattern of the synthesized Praseodymium- Cobaltite nanoparticles. Measurement temperature of 250 C and slit size of 0.6 was set for all measurements. The spectra were recorded in 2θ values ranging from 5.0 to 80.0 with a step increment of 0.005 and count ACS Paragon Plus Environment

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time of 5s. Further, Degree of crystallinity (%)23 of the nanoparticle was calculated from the following equation using the software Origin Pro 2016, Origin Lab Corporation, US. Displayed Equation 1 Percentage Crystallinity =

𝑇𝑜𝑡𝑎𝑙𝐴𝑟𝑒𝑎𝑜𝑓𝑐𝑟𝑦𝑠𝑡𝑎𝑙𝑙𝑖𝑛𝑒𝑐𝑢𝑟𝑣𝑒𝑠 ∗ 100 𝑇𝑜𝑡𝑎𝑙𝑎𝑟𝑒𝑎𝑢𝑛𝑑𝑒𝑟𝑑𝑖𝑓𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛𝑐𝑢𝑟𝑣𝑒

SEM with EDAX Morphology and shape of the as-synthesized Praseodymium-cobaltite nanoparticles was analyzed using ZEISS Sigma 300 Field Emission- Scanning Electron Microscope equipped with Energy Dispersive X-Ray (EDAX) spectrophotometer. The sample was sputtered with gold for 2-3min on stubs and beam of electrons was focused on to the sample and image was obtained. TEM High resolution Transmission Electron Microscope images of the nanoparticles was obtained using Jeol/JEM 2100 HRTEM instrument with a magnification ranging between 2000X – 1500000 X. The resolution and operating voltage of the instrument was 0.23nm and 200kV respectively. The images further confirmed the morphology and size of the synthesized Praseodymium-cobaltite nanoparticles. Isolation of Rat Tail Tendon Collagen Tendons were removed from Rat tail and washed multiple times with cold water, 1:1 diethyl ether and chloroform, methanol and 1% sodium chloride and ground with 0.05M acetic acid. Supernatant was collected by centrifugation followed by precipitation with 5% sodium chloride. Precipitated Type I Collagen was centrifuged and the pellets were collected and dialyzed against 0.05M acetic acid for removing the dissolved salts and was used for further experiments24.

Fabrication of collagen sheets reinforced with Praseodymium-cobaltite nanoparticles


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0.2% collagen was mixed with 1X PBS and the pH was adjusted to 7.2 with 1.25N NaOH for preparing native collagen sheet. Nanoparticle re-inforced collagen sheets were prepared simultaneously by adding suspension of Preaseodymuim- cobaltite nanoparticle in 1XPBS so that the final concentration of nanoparticles was 10, 50 and 100µg/ml. Similarly glutaraldehyde reconstituted collagen sheet was prepared with a final concentration of 50µM. All the preparations were allowed to gel at 370C and washed multiple times with PBS and distilled water for removing un-reacted salts. Conformation studies of collagen FT-IR- ATR Analysis The native, glutaraldehyde reconstituted and nanoparticle re-inforced collagen sheets were subjected to FT-IR- Attenuated analysis. A Jasco FTIR-4700 instrument equipped with ATR facility was used to record the transmittance of Infrared Red rays passed through the collagen sheets. The scanning was carried out from 400-4000cm-1 and the corresponding spectra were recorded. Circular Dichroism Analysis The effect of Praseodymium- cobaltite nanoparticles on conformational integrity of collagen was studied using Jasco J-815 Circular Dichroism Spectropolarimeter. 1ml of 0.4mg/ml collagen was mixed with varying concentrations of nanoparticles and was subjected for analysis in a range of 190-250cm-1at 2000C temperature. 0.05M acetic acid served as blank and native collagen solution was used as control. A positive control with 50µM glutaraldehyde was also set up. Rpn values of the sheets were calculated from the obtained CD spectra by calculating the ratio of positive peak intensity to negative peak intensity. Thermogravimetric Analysis (TGA)


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Thermal stability of the native and re-inforced collagen sheets were analyzed by a TGA Q50 V20.5 Build 30 instrument where the changes in physical and chemical properties of the collagen sheets were analyzed. A known weight of the sample was exposed to increasing temperature ranging from Room Temperature to 8000 C with a constant heating rate of 200C per minute. The percentage reduction in weight of the sample with increase in temperature was recorded. TNBS Assay Tri nitrobenzene sulfonic acid assay was carried out for examining the degree of cross-linking of the cross-linked collagen sheets5. TNBS reacts with unreacted amino group giving a yellow colour solution at the end of the reaction. 2mg each of the re-inforced collagen sheets were incubated in 4% of sodium bicarbonate for 30 minutes followed by the addition of 0.2% 2,4,6Tri nitro benzene sulfonic acid in 4% sodium bicarbonate . After 2hr of incubation at 400C, 6M HCl was added and maintained at 600 C for 30minutes. The solubilized solution was diluted 1:4 with distilled water. The resulted pale yellow solution was read against 335nm using a spectrophotometer. Native collagen sheet served as blank. The reference samples contained appropriate amount of PCNPs to avoid scattering losses.

Displayed Equation 2 Percentage of cross ― linking = 1 ―

Absorbance of cross ― linked collagen sheet ∗ 100 Absorbance of native collagen sheet

Swelling ratio analysis Swelling property of the control and re-inforced collagen sheets were analyzed by immersing a constant amount (2mg) of collagen sheets in 1 ml of 1X PBS25. After fixed intervals of 12hr and


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24hrs, the sheets were removed and excess adhered water was blotted with sterile tissue paper and weight of the sheets were recorded. Displayed Equation 3 Percentage of swelling =

Wet weight of sheets ― Dry weight of sheets ∗ 100 Dry weight of sheets

Displayed Equation 4 Ratio of swelling =

Wet weight of sheets Dry weight of sheets

Proteolytic susceptibility assay Modulation of secretion or activation of extra cellular proteases is a major event in wound repair mechanism26. But the scaffolds meant for wound repair should not get degraded by the activity of such enzymes. For checking the susceptibility of collagen sheets towards proteolytic enzymes, a degradation assay with collagenase enzyme was performed27. Known weight of Collagen sheets were incubated with PBS containing 0.1% of Type 1A Collagenase at 370 C. The weight of the scaffolds was taken at regular intervals after removing the supernatant by centrifugation. The higher the loss in weight corresponds to higher susceptibility towards the enzyme27. Protein adsorption studies Protein adsorption properties of the re-inforced collagen sheets were studied by standard Lowry method28. Bovine serum albumin was used because of its similarity towards Human serum albumin in structure, molecular weight etc. Bovine Serum Albumin (BSA) solution of 200µg mL-1 in PBS was prepared. 1cm2 each of the collagen sheets were immersed in 2ml of BSA solution under shaking at 370 C overnight. The sheets were removed from the solution and the BSA content in the remaining solutions was quantitatively analyzed by Lowry method. The


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reduction in amount of protein in this solution was recorded as the adsorbed protein on the surface of collagen sheets. Native collagen sheet served as control in the study. Hemocompatibility assay Plasma was removed from citrated blood by centrifugation and RBCs were washed several times with 150mM NaCl29. The blood was diluted 1:50 with Phosphate buffered saline, pH 7.4. 200µl of diluted RBCs were incubated with native and re-inforced collagen sheets at 370C for one hour. The intact RBCs were pelleted out by centrifugation and the optical density of the supernatant was read at 540nm. 20% Triton X-100 and Phosphate buffered saline, pH 7.4 was used as positive and negative controls respectively for hemolysis assay. Displayed Equation 5 Hemolysis(%) =

Absorbance of sample – Absorbance of negative control ∗ 100 Absorbance of positive control ― Absorbance of negative control

Whole blood clotting assay Anti-coagulation property of the cross-linked collagen sheets was investigated by anticoagulation assay30. Collagen sheets each of dimension 1cm2 were cut and placed on to 12 well tissue culture plates. Citrated blood of 20µl was poured onto the specimens and 10µl of 0.2mol L-1 CaCl2 solution was dropped into each specimen for initiating blood coagulation. The specimens were then incubated at 370C for 1hr. After incubation, 5ml of water was added onto each well and further incubated for 5minutes at 370C. This water was then collected and centrifuged at 1000g for 1min with a Hermle centrifuge. Subsequently, the supernatant was analyzed for hemoglobin concentration by reading the optical density at 540nm using a Bio Rad ELISA plate reader. All readings were taken in triplicates. Native collagen sheet served as control. Cell Viability assay


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Cell viability of the control-native and cross-linked collagen sheets were performed using MTT assay31 inEA.hy926 endothelial cells. Approximately 20,000- 25,000 cells were seeded on 48 well tissue culture wells plated with native and re-inforced collagen. Cells were incubated till confluency at 370C in 5% CO2 atmosphere and was supplied with Dulbecco’s Modified Eagle’s Medium-High Glucose (DMEM-HG) containing 10% Fetal Bovine Serum (FBS). After attaining confluency, cells were incubated with 0.05% MTT salt [3-(4, 5 dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide] in PBS in dark. Purple colored formazan crystals were formed after 4hrs of incubation and the crystals were dissolved in Dimethylsulfoxide and optical density at 570nm was read using a BioRad ELISA Reader equipment. Displayed Equation 6 Cell viability(%) =

Absorbance of sample ∗ 100 Absorbance of control

Live/Dead Assay Viability of EA.hy926 endothelial cells grown on control-native and fabricated collagen sheets were performed using AO/PI staining32. Native and re-inforced collagen sheets were fabricated on 24 well tissue culture plates. Approximately 30,000- 35,000 cells were seeded on the plate and incubated till confluency at 370C in 5% CO2 atmosphere. The cells were treated with Dulbecco’s Modified Eagle’s Medium-High Glucose (DMEM-HG) containing 10% Fetal Bovine Serum (FBS). 4µM of Acridine orange in PBS, pH 7.4 was added to the cells and incubated for half an hour at 370C. Unreacted acridine orange was washed off and Propidium Iodide (3µM) in PBS, pH 7.4 was added and incubated for 15 minutes at 370C. After incubation, florescence images of the cells were taken with Leica DMi8 microscope. Viable cells appeared in green colour and dead cells appeared in orange to red colour. ROS Detection through DCFH-DA Assay ACS Paragon Plus Environment

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Reactive Oxygen species generation in EA.hy926 endothelial cells grown on native and reinforced collagen sheets were detected by molecular probe, 2, 7 di chloro di hydro fluorescin di acetate (DCFH-DA)33. The cells were treated with 5µM DCFH-DA and incubated half an hour at 370C. The cells were analyzed for green fluorescence intensity through a Leica DMi8 microscope. Increase in intensity of green fluorescence indicates higher ROS formation. Tube formation assay for angiogenesis studies Efficacy of the Praseodymium-cobaltite nanoparticle re-inforced collagen sheets on angiogenesis was studied invitro using EA.hy926 endothelial cells. Cells were grown on native and nanoparticle re-inforced collagen sheets plated on 24 well tissue culture plates. Cells were supplied with Dulbecco’s Modified Eagle’s Medium-High Glucose (DMEM-HG) containing 10% Fetal Bovine Serum (FBS) and allowed to grow up to 24hrs on the collagen sheets. Formation of endothelial tubes was observed through a Leica DMi8 phase contrast microscope34. The images were quantified for Total tubule length using AngioSys 2.0 software, Cellworks, UK35. Processing of the images prior to quantification was done with Adobe Photoshop, Adobe Inc., United States and Picasa3, Google, Inc. Aortic arch assay for angiogenesis studies Aorta was carefully removed from 11th Day fertilized egg embryos36 and placed in serum free Dulbecco’s Modified Eagle’s Medium-High Glucose (DMEM-HG) after multiple washes with PBS pH 7.4. The aortic arches was trimmed and cut into 1mm pieces and placed into 24 well tissue culture plate containing native and Praseodymium-cobaltite nanoparticle cross-linked collagen sheets. The aortic arches were maintained in a 5% CO2 incubator at 370C. Images of sprouting of endothelial cells were captured once in 2 days with a Leica DMi8 microscope. Fluorescent images of the sprouts were captured by adding molecular probe, Calcein AM (3µM). Stem cell differentiation studies ACS Paragon Plus Environment

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Human Wharton’s Jelly mesechymal stem cells (HWJMSC) were procured from HiMedia Labs, India for studying the effect of cross-linked collagen sheets on stem cell differentiation. The cells (20,000-25,000) were seeded on 24 well tissue culture plate coated with fabricated collagen sheets. The cells were allowed to grow in a Binder CO2 incubator set at 370C for 1-3days. The cells were supplied with Human Wharton’s Jelly Expansion Media containing Fetal Bovine serum and essential antibiotics. The morphology changes resembling endothelial cells were analyzed with a Leica DMi8 phase contrast microscope. Invivo wound healing assay Wound healing efficacy of PCNP (Praseodymium-cobaltite nanoparticles) cross-linked collagen sheets were understood with invivo open excision healing assay37 in male wistar albino rats after getting approval from Institutional Animal Ethical Committee at CSIR- Central Leather Research Institute, Chennai. Rats weighing 100-140g of 6-8 weeks old were housed in transparent polycarbonate cages and were acclimatized for a week in animal facility conditions. Temperature and humidity of the animal house facility was maintained at 200 ±3ºC and 50% respectively throughout the studies. The rats were provided with pellet feed and unlimited supply of water. Animal facility was maintained 12hrs in light and 12hrs in dark. Animals were shaved on the dorsal side 24hrs prior to the creation of wounds. Rats were anesthetized by IP administration of Ketamine +Xylazine in a dosage of 80mg/kg +10mg/kg38 and an open excision wound of size 2X2cm was made by carefully cutting the dorsal skin of the rats after disinfection. Collagen sheets cross-linked by PCNPs of different concentrations were applied onto the wound site and the area was covered by absorbent sterile gauze on test group animals. Control groups were applied with native collagen sheet and untreated saline control rats were also grouped. The dressings were changed once in 4days after cleaning the wound area with saline. The wound area was traced on to coordinate paper each time during changing of dressings. The total number of ACS Paragon Plus Environment

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grids in the area was counted manually and it was recorded as wound size of that particular day. Macro photographs of wound size were also captured simultaneously. The percentage reduction in wound size was calculated from the traced coordinate paper using the formula. Displayed Equation 7 Cn =

[

]

So ― Sn × 100 So

where, Cn is the percentage reduction in wound size on 3rd,7th,11th,15th and 17th day. So is the wound size on 0th day and Sn is the wound size on 3rd, 7th, 11th, 15th and 17th day. Statistical analysis of the reduction in wound area was done by One-way ANOVA followed by Tukey’s Multiple comparisons test using Graph Pad Prism 8 software, USA. Histological Analysis One animal from each group were sacrificed on Day 15 for histological analysis. Excised skin was embedded in paraffin wax after fixing in 10% formaldehyde. Masson’s Trichrome39 staining was performed for investigating collagen deposition by trimming 5µm of tissue and sequential deparaffinization

followed

by

rehydration.

Staining

with

weigert’s

hematoxylin,

phosphomolybdic acid, aniline blue, acetic acid and ethanol was performed according to standard procedures. Images of the stained tissue for collagen deposition, hair follicle formation and blood vessel formation were captured by Leica DMi8 phase contrast microscope. Results and discussion Synthesis of Praseodymium-cobaltite nanoparticles (PCNP) Praseodymium-cobaltite oxide nanoparticles were synthesized by sol-gel method with 1: 0.7 ratio of praseodymium nitrate and cobalt nitrate as precursors respectively with 0.04M citrate as reducing agent. The reduction of precursors took place at 900C with the formation of a gel. The subsequent calcination of the gel at 8000C yielded PCNPs appearing in black colour. Centrifuge


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washing with acetone, ethanol and nanopure water removed unreacted precursors, reducing agent as well as larger sized particles. Characterization of nanoparticles Particle size analysis The size and dispersity of the as-synthesized Praseodymium-cobaltite nanoparticles was analyzed using Dynamic Light Scattering technique. A single peak showing an average size range of 153nm was obtained for the PCNPs in aqueous medium. A good Polydispersity Index (PDI) of 0.097 was obtained. The results show that the particles are in nano size range. The size obtained through DLS analysis is inclusive of the size of surrounding water layer that covers the particles (hydrodynamic size)40. Thus the actual size of the nanoparticle will be less than the size obtained through Dynamic Light Scattering technique. Polydispersity index (PDI) is a measure of monodispersity of samples in solution. Lower PDI index infers to the monodisperse nature of the nanoparticles. PDI values greater than 0.4 indicate highly polydisperse samples. Values between 0.4 and 0.1 are accepted as moderately polydisperse samples with decrease in the value indicating higher monodispersity. A value less than 0.1 are rarely observed and show highest monodispersity40. The lower PDI of 0.097 obtained for Praseodymium-cobaltite nanoparticle shows that the particles doesn’t forms clumps and are dispersed evenly in solution. Powder X-RD The crystallinity of the Praseodymium – cobaltite nanoparticles was explored using powder XRay Diffraction Technique(Figure 1). As Dynamic Light Scattering provides only indicative size of the particles40, the confirmation of sizes of nanoparticles has to be investigated with other techniques. The XRD pattern in the figure corresponds to the product obtained by thermal reaction of Pr (NO3)3.6H20, 0.07M and Co (NO3)2. 6H20 in the ratio 1: 0.7 in the presence of ACS Paragon Plus Environment

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0.4M citric acid. The XRD patterns analyzed in reveal that the synthesized PraseodymiumCobaltite nanoparticles are in crystalline form. The spectral analysis with Rigaku PDXL integrated X-ray powder diffraction software showed that the peaks of Pr6O11at 2θ values of 28.2, 46.95 and 55.6 corresponds to Praseodymium oxide with hkl values of (1 1 1), (2 2 0) and (3 1 1) respectively. The data matches with the number 60329 in ICDD date base showing cubic phase with space group Fm-3m. The sharp peaks indicate highly crystalline nature of the synthesized PCNPs. The peaks obtained for Cobalt oxide phase of the PCNPs was of less intensity compared to Praseodymium phase of the nanoparticles. It may be attributed to the less weight percentage of total Cobalt oxide nanoparticles formed after the reaction. EDAX data supports this assumption. The small peaks at 2θ values of 19.01, 36.55 and sharp peak at 59.56 corresponds to hkl values of (111), (311), and (511) respectively. JCPDS Card no. 43-1003 matches with these peaks and proves that the Cobalt oxide nanoparticles exist in cubic Co3O4 phase41. Percentage Crystallinity was calculated from the X-RD pattern. Degree of crystallinity is the ratio of crystalline phase to amorphous phase in a particular sample under study42. The results show 87% of crystallinity for the as-synthesized Praseodymium-cobaltite nanoparticles. Thus, it was confirmed that the PCNP are highly crystalline in nature. SEM with EDAX The morphology and shape are important tools for the characterization of nanoparticles43. Scanning electron Microscope observation showed the morphology of the nanoparticles as spherical (Figure 2.a). A uniform distribution in size with slight aggregation of particles was observed. The elemental composition of the synthesized nanoparticles was analyzed through an EDAX Spectrum43,44. Electromagnetic emission of each atom appears as unique peaks in spectrum. EDAX spectrum of ACS Paragon Plus Environment

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Praseodymium- Cobaltite nanoparticles (PCNPs) showed 71% of Praseodymium, 17% of Cobalt and 12% of Oxygen atoms in the sample under the study (Figure 2.b). TEM The size and morphology of the synthesized Praseodymium- Cobaltite nanoparticles was further confirmed by Transmission Electron Microscope analysis43,44. Results obtained by X-RD measurement are confirmed in the TEM analysis and size of particles was observed to be in the range between 100 and 150nm (Figure 3). The shape of the nanoparticles obtained through TEM analysis is in concordance with the images of SEM Analysis showing spherical morphology. The image shows that some of the nanoparticles exist as caged one around the other. A darker bulk portion of the nanoparticle is covered by a lighter layer as seen from the Figure 3. It is assumed that the darker bulk and lighter covering layer may be Praseodymium and Cobalt respectively. Conformation studies of collagen sheets FT-IR- ATR Analysis The conformational integrity of collagen after cross-linking with PCNPs was studied with FTIR-ATR spectroscopic analysis. The characteristic vibrational spectral bands of collagen were recorded. The FTIR spectral bands of native collagen, glutaraldehyde cross-linked collagen and collagen cross-linked with 10µg/ml, 50µg/ml and 100µg/ml of PCNPs are shown in the Figure 4. The positions and nature of the characteristic bands was same in all the five samples under study. The peaks observed at 1628cm-1 corresponded to amide I vibrations of carbonyl (C=O) groups and peak at 1546cm-1clearly denoted the presence of amide II (N-H) vibrations of collagen. Amide III vibrations was observed by the presence of peak at 1234cm-1 demonstrating the triple helical structure of collagen45,46 Amide A bands was observed at 3293cm-1 and Amide B bands corresponding to asymmetrical stretching of -CH2 was noted at both 2934cm-1and 3293cm-1 45,46. The increased intensity of peaks at 1030cm-1 in PCNPs cross-linked collagen sheets might be ACS Paragon Plus Environment

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showing the bidentate carbonate bounded with Pr cation47. No larger shifts in the spectral bands were observed in the results. The results indicated that the cross-linking with PCNPs has not caused any alterations to the triple helical structural integrity of collagen. The integrity of collagen triple helix was evaluated by the analysis of ratios of absorbance band at 1235cm-1 and 1450cm-1. Ratios close to 1 demonstrates integrity of Type I collagen and ratios close to 0.5 indicates denaturation48. The relative 1235cm-1/1450cm-1ratio of native, glutaraldehyde reconstituted and PCNP cross-linked collagen showed values between 0.9-1 indicating the intact triple helical structure of collagen. Circular Dichroism The impact of incorporation of PCNPs on three dimensional structure of collagen molecules was characterized by Circular Dichroism spectroscopic analysis. Native collagen exhibits unique CD spectra with small positive peak around 220nm, a cross-over around 214nm and a large negative peak around 197nm49. The spectra didn’t showed large shifts in any of the positive, cross-over or negative peaks showing that the structural integrity of collagen is preserved after cross-linking with PCNPs (Figure 5), (Table 1). Rpn value is the ratio of positive peak intensity to negative peak intensity49. The Rpn ratios and CD spectrum of PCNPs cross-linked collagen compared with native and glutaraldehyde crosslinked collagen is shown in the Table 1 below. The Rpn values of native collagen and crosslinked collagen doesn’t show much change. Thus, it was confirmed that the triple helical conformation of collagen molecules is intact following cross-linking with different concentrations of PCNPs even though intensities of peaks has slightly changed. Thermogravimetric Analysis (TGA) The changes in thermal properties brought about by the addition of PCNPs into collagen sheets were studied by thermogravimetric analysis. The rate of change in percentage weight of native as ACS Paragon Plus Environment

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well as cross-linked collagen sheets were analyzed as a function of temperature (Figure 6). Thermal degradation of collagen happened in three steps46. The first phase between 75-1000C corresponded to the removal of physically adsorbed water molecules46 with weight loss of 13%. The second transition at 2420C corresponded to the removal of structural water46 with weight loss of 4%. A shift towards higher transition temperature was observed with PCNP cross-linked collagen. The second transition happened at 2420C for native collagen and 2600C for gultaraldehyde cross-linked collagen with percentage weight loss of 4%. However, the second transition of PCNP cross-linked collagen sheets occurred only between 2700C- 2900C. The weight loss of PCNP cross-linked collagen sheets at this stage was slightly higher than that of native collagen sheet which might be featured to the increase in water holding capacity of the cross-linked scaffolds compared to native collagen scaffolds. Swelling ratio analysis results are in complement with these findings. The third degradation phase of native collagen ranged between 4200C to 4500C but for glutaraldehyde and PCNP cross-linked collagen it ranged between 4300C to 4900C with the least weight loss observed with collagen sheet cross-linked with 50µg/ml and 100µg/ml of PCNPs. The reduction in weight loss was due to the increased formation of inter-fibrillar cross-linking with PCNPs. At the endpoint of 8000C, 85% of native collagen got degraded while 18% of glutaraldehyde cross-linked collagen remained. A momentous increase in the mass of residues of PCNP cross-linked collagen sheet with increased concentration of PCNPs was noted at the endpoint temperature. 20%, 22% and 32% of residues was left over for collagen cross-linked with 10µg/ml, 50µg/ml and 100µg/ml PCNPs respectively at 8000C. From the results it is confirmed that incorporation of PCNPs improved the thermal stability of the collagen scaffolds. This stability might be attained by the introduction of more number of covalent bonds between the collagen fibrils in PCNP cross-linked collagen sheets. TNBS assay ACS Paragon Plus Environment

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The degree of cross-linking in collagen sheets cross-linked with PCNPs was compared with the collagen control and glutaraldehyde cross-linked collagen sheets. The free amino groups of lysine present in the samples reacted with TNBS reagent and the OD values at 345nm were recorded. The absorbance and amount of free amino groups are directly proportional5.The percentage cross-linking was observed to be higher in nanoparticle reinforced collagen sheets than in native collagen sheet. Glutaraldehyde reinforced collagen sheets which served as the positive control showed the highest percentage of cross-linking. The percentage of cross-linking obtained for 10µg/ml, 50µg/ml and 100µg/ml PCNP reinforced collagen scaffolds were 30%, 40% and 60% respectively (Table 2). The glutaraldehyde crosslinked collagen sheets showed a percentage cross-linking of 70%. The results obtained showed that with increase in concentration of nanoparticle, the amount of free amino groups got reduced and yielded better cross-linking. It is reported that the nanoparticles forms nucleation sites in collagen triple helix and initiates cross-linking9. Thus we assume that, with increase in amount of nanoparticles used, more nucleation sites have formed for the collagen molecules to get crosslinked. This may be the reason for obtaining 2 fold increase of cross-linking percentage in collagen sheet incorporated with 100µg/ml of PCNP than that with 10µg/ml of PCNP. Swelling ratio analysis The water retention capacity of the PCNP cross-linked collagen sheets were analyzed by a swelling-ratio analysis. Uptake of PBS by the sheets was recorded as increase in weight percentage of the samples in different intervals till reaching saturation. A decrease in swelling of the collagen sheets was observed with increase in concentration of the nanoparticles used. A wound dressing with the capacity to retain moisture can absorb wound fluids exudating from the damaged tissue area effectively. These wound fluids are sources of microbes and its proper removal from the wound site can be done with the applied dressing itself50. The most water ACS Paragon Plus Environment

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retention was obtained with collagen sheet cross-linked with PCNP (10µg/ml). The results are summarized in Table.3. No increase in swelling was observed for the samples after 24hrs and it attained a steady state in swelling by this time. It is proposed that the water molecules get trapped into the pores present on the scaffold50. Native collagen sheet showed less water retention compared to cross-linked collagen sheets. This can be attributed to the reduced mechanical stability of native collagen in moist environment due to poor crosslinking when compared with nanoparticle cross linked collagen. The decrease in water retention with increase in concentration of PCNP used can be attributed to the slight concentration dependent decrease in hygroscopic nature of the nanoparticles as well as decrease in pore size of PCNP cross-linked collagen sheets with increase in concentration of PCNPs. It is expected that this decrease in swelling ratio will not affect the biological properties of the sheets and the same is confirmed in further studies. Proteolytic susceptibility assay Wound microenvironment is always rich with extra cellular proteases as a response against the microbial infection26. The dressing materials meant for healing the wounds are always susceptible to the activity of protease enzymes and degradation of the scaffolds can happen frequently27. Native collagen sheets are more susceptible to such degradation27. It is proposed that the cross-linking of collagen can reduce the proteolytic susceptibility towards extra cellular proteases27. Collagenase is a major enzyme secreted by various cells51 causing the degradation of collagen leading to delayed wound healing in pathological conditions. Figure.7 shows the proteolytic susceptibility assay results of PCNP cross-linked collagen sheets. The susceptibility of PCNP cross-linked collagen sheets were analyzed by immersing the scaffolds in Collagenase enzyme at 370C overnight. The results showed that with increase in concentration of PCNPs used, susceptibility of collagen sheets towards the enzyme got reduced. ACS Paragon Plus Environment

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The results were recorded as the reduction in weight of samples after overnight incubation at 370C. The reduction in collagenase susceptibility of PCNP cross-linked collagen sheets with increase in concentration of PCNP used might be attributed to the mild inhibitory effect of PCNP on collagenase enzyme49. Further, it would be justified with the phenomenon of bringing about of increased number of collagen cross-links when the collagen is incorporated with PCNPs in a concentration dependent manner52. The results showed that the PCNP cross-linked collagen sheets are promising for use as wound dressing material. Protein adsorption studies Protein adsorption and complement protein activation occurs when a biomaterial is introduced into the body. These complement proteins are agents of inflammation and thus they need to be regulated for expediting the wound healing process53. Albumin is the most abundant protein in blood and thus it was taken as a model protein for investigating the protein adsorption behavior of PCNP cross-linked collagen sheets53. The PCNP cross-linked collagen scaffolds was analyzed for albumin adsorption by immersing them in 200µg mL-1 of BSA in PBS. A less adsorption of albumin with increasing concentration of PCNPs used was obtained through spectrophotometric observation at 280nm. Native collagen sheet showed the highest protein adsorption of 83.1 µg mL-1. Protein adsorption of 57.6µg mL-1, 55.8µg mL-1 and 48.8µg mL-1 was obtained for collagen sheets cross-linked with PCNP(10µg/ml), PCNP(50µg/ml) and PCNP(100µg/ml) respectively. Less albumin adsorption is a desirable property for a wound dressing material as the adsorbed protein can cause further activation of enzymes such as elastase causing degradation of elastin53. Degradation of elastin leads to delaying of wound healing54. The less albumin adsorption of PCNP cross-linked collagen sheets can be attributed to the hydrophilic property of the PCNP cross-linked collagen sheets. Pore size of the collagen sheets might be getting reduced with reinforcement with PCNPs. ACS Paragon Plus Environment

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Hence, the BSA will not be able to fit onto the pores leading to reduced adsorption on PCNP cross-linked collagen sheets than that of native collagen sheets. Hemocompatibility Assay The wound dressings meant for tissue engineering should be compatible to blood as it will be in frequent contact with the blood around the wound environment. Hemocompatibility of the PCNP cross-linked collagen scaffolds in citrated human blood was observed byhemolytic assay. The results showed good hemocompatibility for PCNP cross-linked collagen scaffolds. The rupture of RBCs was negligible during the incubation time. Hemolysis percentage of the collagen scaffolds cross-linked with different concentrations of (10µg/ml, 50µg/ml and 100µg/ml) PCNPs ranges between 0.14 - 0.28%. There was no significant difference in hemolytic percentage for the native collagen scaffolds. The slight increase in hemolysis with increase in PCNPs might be attributed to the minor increase in hydrophobicity of collagen sheet cross-linked with 100µg/ml of PCNPs. Substances exhibiting hemolysis less than 5% are considered to be hemocompatible55. Hence, the hemolysis results confirm the hemocompatible nature of PCNP cross-linked sheets as shown in Table.4. Whole blood clotting assay Responses of PCNP cross-linked collagen sheets on coagulation of blood were analyzed by whole blood clotting assay. The optical density of the hemoglobin solution read at 540nm showed an increase in OD value with increase in concentration of nanoparticles (Figure 8) for PCNP cross-linked collagen sheets. The increase in OD values indicates a less amount of hemoglobin/RBCs was used in the clot formation56. Clotting observed with collagen cross-linked with 50µg/ml and 100µg/ml PCNPs were fairly less when compared to native collagen sheet. Native collagen sheet and 10µg/ml PCNP cross-linked collagen sheet didn’t show much difference in OD values. Therefore, PCNP cross-linked collagen sheets do not make the blood to ACS Paragon Plus Environment

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clot and is safe to be used as a biomaterial. Along with hemolysis results, the anticoagulation assay too confirms the hemocompatibility of PCNP cross-linked collagen sheets. The reduction in blood-clotting ability of PCNP cross-linked collagen sheets might be due to the denaturation of coagulation proteins brought about by the therapeutic property of Praseodymium-cobaltite nanoparticles30,56. Cell Viability assay One of the main conditions a biomaterial should possess is compatibility to the host body. It should not elicit any unendurable toxic effects to the body1,57,58. The biocompatibility of PCNP cross-linked collagen sheets on blood was analyzed by hemocompatibility assay and has given good positive results. Further, the toxic effects of wound dressing materials on human cells was studied by cell viability assay using MTT Assay [3-(4, 5 dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide] in EA.hy926 cells. Figure 9 shows the cytocompatibility assay results of PCNP cross-linked collagen sheets. Collagen cross-linked with 10µg/ml, 50µg/ml and 100µg/ml of PCNPs shows percentage cell viability of 99.7%, 94.3% and 91.44% respectively. The calculations were done by keeping the percentage cell viability of native collagen sheet as 100%. The results showed that the collagen sheets are compatible to the cell line under study. The results may be attributed to synergestic therapeutic effect of Praseodymium-cobaltite nanoparticles as well as the hydrophilicity and porous of the sheets50,59. Porous nature of the sheets as observed from swelling ratio analysis allows the exchange of nutrients and gases required for the nourishment of cells leading to improved cell attachment and growth60. The findings are in agreement with swelling ratio analysis and hemocompatibility results. Live/Dead Assay


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Acridine orange/ Propidium iodide staining was done in EA.hy926 endothelial cell line treated with PCNP cross-linked collagen sheets as a confirmation for cell viability assay. The images captured after prescribed incubation of cells with the dual dyes doesn’t show any toxicity of PCNP cross-linked collagen sheets. Negligible number of cells was emitting orange-red fluorescence relating to early and late apoptosis with the collagen sheet containing 100µg/ml PCNPs32. The results are shown as microphotographs in Figure 10. Collagen sheets cross-linked with 10µg/ml and 50µg/ml PCNPs didn’t show any fluorescence corresponding to apoptosis. Little difference was observed in results between PCNP cross-linked collagen sheets and native collagen sheet. The toxicity results are in concordance with the results obtained from MTT Assay. The results suggest improved growth and perfusion of cells61 on the sheets even with higher concentrations of the nanoparticles used. Thus it is confirmed that the PCNP cross-linked collagen sheets provides necessary microenvironment for the endothelial cells to proliferate without imparting any significant toxic effects. ROS Detection through DCFH-DA Assay Intra cellular ROS production elicited by PCNP cross-linked collagen scaffolds was qualitatively investigated by 2, 7 di chloro di hydro fluorescin di acetate (DCFH-DA) assay. The green fluorescence corresponding to intracellular ROS generation33 was minimally observed with native collagen sheet as well as collagen scaffolds fabricated with 10µg and 50 µg of PCNPs (Figure 11). Low levels of fluorescence were observed with 100µg/ml PCNP cross-linked collagen scaffolds. The low level expression of ROS corresponds to the normal metabolic functions of the cell62. Reports are available demonstrating the ability of mild levels of ROS in promoting angiogenesis63. This property might be beneficial in hastening the wound healing invivo63, which is discussed in the later part of this article. The ability to elicits low level of ROS in endothelial ACS Paragon Plus Environment

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cells by the PCNP cross-linked collagen sheets is quite promising in tissue regeneration process. This property of the sheets can be featured to its high biocompatible nature which was confirmed via various assays as mentioned earlier. Tube formation assay for angiogenesis studies A biomaterial with pro-angiogenic effect can facilitate a faster wound healing in damaged tissues63. Investigation into the pro-angiogenic property of PCNP cross-linked collagen sheets were done with invitro tube formation assay. The quantification of captured images showed that the formation of endothelial tubes was enhanced by the addition of PCNPs in the collagen matrix (Figure 12.a). A dose dependent increase in the formation of tube like network was observed after 8hrs of treatment. With increase in concentration of PCNPs used, total tubule length of endothelial tubes grown on cross-linked collagen sheet shows gradual increase (Figure 12.b). The PCNP cross-linked collagen sheets as described in MTT Assay is showing good compatibility to endothelial cell lines. Angiogenesis occurs through a sequel of events predominantly involving the division and proliferation of endothelial cells

64.

The therapeutic

effects of the PCNPs along with the collagen have promoted the growth and proliferation of the endothelial cells and the cells got differentiated to form tubes demonstrating formation of tubular capillaries. It is proposed that the PCNPs are triggering the production of Vascular growth factors guiding the formation of endothelial tubes 64,65. Aortic arch assay for angiogenesis studies Pro-angiogenic effect of PCNP cross-linked collagen sheets was confirmed by the endothelial tube formation assay. Invivo analysis of angiogenic effect of the collagen sheets are further studied by aortic arch assay. Sprout formation from the aortic arches are evidences for the proangiogenic effect of the sample under the study

36,66.

Pro-angiogenic effect is an inevitable

property, a biomaterial should possess 64. Chances of rejections by the host systems are high for a ACS Paragon Plus Environment

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biomaterial having no angiogenic effect. Sprouting from the aortic arches started appearing from the 4th day of experiment and fluorescence micro-images of the assay was captured at the end of 14th day by the addition of fluorescent probe, Calcein AM. Figure 13 shows the fluorescent images of the capillary networks appeared on the native and PCNP cross-linked collagen scaffolds. Aortic arches placed on to the collagen sheets incorporated with PCNPs at all concentrations (10µg/ml, 50µg/ml and 100µg/ml) showed good fluorescence. No appreciable sprouting was observed with aortic arches placed on native collagen sheet. Quantified results showing total tubule length of sprout formation are displayed in Figure 14. This assay confirms the pro-angiogenic therapeutic effect of Praseodymium-cobaltite nanoparticles. Blood vessels are composed of three distinct layers and these layers consist of several kinds of cells other than endothelial cells such as smooth muscle cells, fibroblasts, and perivascular nerves64. The compatibility and proliferation potency of PCNP cross-linked collagen sheets on endothelial cells were proved by tube formation assay. Further, the aortic arch assay confirmed that the cross-linked collagen sheets are compatible as well as possess growth promotive effect on other cells involved in the formation of blood vessels. This angiogenic effect of the collagen sheets is advantageous in the orchestration of wound healing cascade67. The improved angiogenesis impeded by the cross-linked collagen sheets fastened the tissue granulation by improving re-epithelialisation and tissue remodeling at the final stages of wound healing26,67. Stem cell differentiation studies Differentiation of stem cells into endothelial cells is a promising property which a biomaterial may possess68. The effect of PCNP cross-linked collagen sheets on differentiation of mesechymal stem cells into endothelial cells was studied. Morphological changes of Human Wharton’s Jelly mesechymal stem cells (HWJMSC) resembling endothelial cells were observed ACS Paragon Plus Environment

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for the cells growing on the biomaterial in all the concentrations of PCNPs under study viz. 10µg/ml, 50µg/ml and 100µg/ml (Figure 15.a).The total tubule length of the differentiated cells is shown in Figure 15.b. Endothelial tube like morphology during the end of study was observed for HWJMSCs grown on PCNP cross-linked collagen sheet. Total tubule length of these tubes showed a concentration dependent increase in the amount of PCNPs used with collagen sheet cross-linked with 100µg/ml of PCNPs giving the maximum. As already seen from the invitro studies, Praseodymiumcobaltite cross-linked collagen sheets are found to be endowed with pro-angiogenic effect. This effect might be because of the therapeutic potential of the nanoparticles causing an increased production of Vascular Endothelial growth factor (VEGF)64,65.

Reports are already been

available showing the effectiveness of mesenchymal stem cells in enhancement of wound healing process39,69,70. This is achieved via differentiation of thus introduced mesenchymal stem cells into endothelial or fibroblast cells at the wound area causing induction of angiogenesis and increased migration of fibroblasts39,69,70. Thus the PCNP cross-linked collagen sheet which proved to have the ability to differentiate mesenchymal stem cells can be exploited further in soft tissue engineering field. Invivo wound healing assay The invitro studies on proteolytic susceptibility, biocompatibility and pro-angiogenic properties showed positive results with PCNP cross-linked collagen sheets compared to control sheets. Further investigation into the wound healing activity of the cross-linked scaffolds was done invivo on 6-8week old wistar albino male rats under standard animal facility conditions37. Collagen sheets cross-linked with 10µg/ml, 50µg/ml and 100µg/ml of PCNPs along with control native collagen was investigated for their efficacy in wound healing process. Results from the untreated control group with application of saline alone onto the wound site were compared with ACS Paragon Plus Environment

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percentage wound reduction from the cross-linked collagen sheet treated test group. Figure 16 shows the microphotographs of open excision wounds taken in different time intervals between 0-17days. Percentage reduction in wound size was calculated quantitatively and the results are shown in Figure 17. Complete wound closure was observed with all concentrations of PCNP cross-linked collagen sheets by day 17. 70%, 72% and 91% reduction in wound area was observed on rats treated with collagen sheets cross-linked by 10µg/ml, 50µg/ml and 100µg/ml of PCNP respectively on 11th day itself while native collagen control rats showed only 64% wound area reduction. The results of the statistical analysis using One-way ANOVA are summarized in Table S1 as supplementary information. All the test samples (Col+PCNP10, Col+PCNP50 and Col+PCNP100) show significant difference against saline control for all the days. The test compounds showed significant differences between each other except on Day 17. The test samples caused the wounds to close by Day 17 due its therapeutic property. Hence there was no significant difference within various treated groups. Col+PCNP10 showed no significant differences against native collagen on until day 11th. Col+PCNP100 showed significance difference in wound reduction against native collagen and saline control on all days. Col+PCNP100was observed to be superior to Col+PCNP10 and Col+PCNP50 treated rats. Thus the results confirmed that Col+PCNP100 can be used as wound healing agent for cutaneous wounds effectively. The faster rate of wound healing cascade may have been accomplished through reduced inflammatory effect accompanied by angiogenic and reduced proteolytic susceptibility effects39,60 of the PCNP cross-linked collagen sheets as observed from other assays carried out in this study.

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Masson’s Trichrome Staining of the 15th Day excised tissue from the skin of rats under the study shows results consistent with reduction percentage of wound area. Rats treated with collagen cross-linked with 10µg/ml, 50µg/ml and 100µg/ml of PCNP showed increased collagen deposition than that of saline and native collagen sheet treated rat skin. Figure 18 showed more ordered arrangement of collagen fibrils (stained in blue colour) in test sample treated rat skins. Among these test samples, collagen cross-linked with 100µg/ml of PCNP showed higher deposition of collagen as well as hair follicle and blood vessel formation (stained in red colour). The results clearly showed evidence of re-epithelialization and increased healing of wounds in nanoparticle cross linked collagen. Conclusions The study involved the re-inforcement of collagen scaffolds for minimizing its drawbacks while used as a biomaterial. Praseodymium-cobaltite nanoparticles have efficiently shown to be a good cross-linker for collagen by increasing its physicochemical and biochemical properties. The results showed that the microenvironment surrounding the mammalian cells was nourished by the Praseodymium-cobaltite nano-formulation released by the collagen sheets and acted therapeutically for improving pro-angiogenic effects. The biological benefits of praseodymiumcobaltite cross-linked collagen sheets such as biocompatibility, reduced susceptibility to proteolytic degradation and pro-angiogenic effects has its use for wound healing applications. Invivo study in rat models confirmed the promising effect of the cross-linked collagen sheets over tissue engineering applications. Thus, this study paves the way for identifying better biomaterials based on rare earth metal nanoparticles for tissue regeneration applications.


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author *Manikantan

Syamala Kiran

Senior Scientist, Assistant Professor, Academy of Scientific and Innovative Research, Council of Scientific and Industrial Research- CLRI, Adyar, Chennai-20, Tamilnadu, India Email: [email protected] and [email protected] Conflicts of interest There are no conflicts to declare. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding sources The first author acknowledges Department of Science and Technology, Govt.of India for providing financial assistance in the form of INSPIRE fellowship (IF160615). Acknowledgements We are grateful to thank The Director, CSIR-Central Leather Research Institute, Chennai, India for providing the necessary facilities for carrying out the analyses. This work was carried out as a


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part of the PhD research work under University of Madras, Chennai, India. We thank DSTSophisticated Analytical Instruments Facility, STIC, Kochi, India for their help in carrying out electron microscopic analyses. Supporting Information Stem cell differentiation property of PCNP cross-linked collagen sheets (Figure S1). Statistical analysis of wound healing assay in albino wistar rats (Table S1). Abbreviations SEM- Scanning Electron Microscope, EDAX- Energy Dispersive X-Ray Spectroscopy, TEMTransmission Electron Microscope, FT-IR-ATR- Fourier Transform-Infra Red- Attenuated Total Reflectance ,TNBS- Trinitrobenzenesulfonic acid References (1)

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(13) Nethi, S. K.; Barui, A. K.; Bollu, V. S.; Rao, B. R.; Patra, C. R. Pro-Angiogenic Properties of Terbium Hydroxide Nanorods: Molecular Mechanisms and Therapeutic Applications in Wound Healing. ACS Biomater Sci Eng. 2017, 3, 3635–45. (14) Peng, L.; Weiying, Z.; Xi, L.; Yi, L. Structural Basis for the Biological Effects of Pr(III) Ions: Alteration of Cell Membrane Permeability. Biol Trace Elem Res. 2007, 120, 141–147. (15) Basile, A. C.; Hanada, S.; Sertie, J. A.; Oga, S. Anti-Inflammatory Effects of Praseodymium, Gadolinium and Ytterbium Chlorides. J Pharmacobiodyn. 1984, 7, 94–100. (16)Andiappan, K.; Sanmugam, A.; Deivanayagam, E.; Karuppasamy, K.; Kim, H.-S.; Vikraman, D. In Vitro Cytotoxicity Activity of Novel Schiff Base Ligand–Lanthanide Complexes. Sci. Rep. 2018, 8, 1-12. (17)Angrisani, N.; Reifenrath, J.; Zimmermann, F.; Eifler, R.; Meyer-Lindenberg, A.; VanoHerrera, K.; Vogt, C. Biocompatibility and Degradation of LAE442-Based Magnesium Alloys after Implantation of up to 3.5years in a Rabbit Model. Acta Biomater. 2016, 44, 355–365. (18) Zhang, M.; Ma, S.; Xu, K.; Chu, P. K. Corrosion Resistance of Praseodymium-IonImplanted TiN Coatings in Blood and Cytocompatibility with Vascular Endothelial Cells. Vacuum. 2015, 117, 73–80. (19) Tanaka, T.; Kojima, I.; Ohse, T.; Ingelfinger, J. R.; Adler, S.; Fujita, T.; Nangaku, M. Cobalt Promotes Angiogenesis via Hypoxia-Inducible Factor and Protects Tubulointerstitium in the Remnant Kidney Model. Lab Invest. 2005, 85, 1292–1307.


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FIGURES

Figure 1.Powder X-Ray Diffraction pattern of Praseodymium-cobaltite nanoparticles

Figure 2.(a)-Scanning Electron Microscope image of Praseodymium-cobaltite nanoparticles showing spherical morphology. Scale bar = 200nm, (b)-EDAX elemental analysis spectrum of Praseodymium-cobaltite nanoparticles


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Figure

3.Transmission

Electron

Microscope

image

of

Praseodymium-cobaltite

nanoparticlesshowing a size range between 100 and 150nm with spherical shape. Scale bar = 20nm

Figure 4.FT-IR-ATR spectra of a. native collagen sheet b.glutaraldehyde cross-linked collagen sheet c. collagen cross-linked with PCNPs 10µg/ml d. 50µg/ml and e. 100µg/ml


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Figure 5.CD spectra of Native, glutaraldehyde and PCNP cross-linked collagen sheets showing conservation of triple helical structure of collagen molecules

Figure 6.TGA curves of (a)-Native and glutaraldehyde cross-

linked collagen sheets

(b)-

PCNP cross-linked collagen sheets


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Figure 7.Proteolytic degradation assay results showing reduced susceptibility of PCNP crosslinked collagen sheets towards collagenase enzyme

Figure 8.Optical density readings of hemoglobin solution obtained by lysing the unbound RBCs after incubation of whole blood on Native collagen sheet, Collagen sheet cross-linked with PCNPs 10µg/ml, 50µg/ml and 100µg/ml


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Figure 9. (a)- Phase contrast microphotographs of MTT Assay with EA.hy926 cells grown on A. Native collagen sheet,Collagen sheet cross-linked with B. PCNPs (10µg/ml), C. PCNPs (50µg/ml), D. PCNPs (100µg/ml) (b)- MTT Assay results of Native and PCNP cross-linked collagen sheets showing good cytocompatibility of PCNP cross-linked collagen sheets

Figure 10.Fluorescence microphotographs of AO/PI staining of EA.hy926 cells grown on A.Native collagen sheet, B. Collagen sheet cross-linked with PCNPs (10µg/ml), C. Collagen sheet cross-linked with PCNPs (50µg/ml), D. Collagen sheet cross-linked with PCNPs (100µg/ml)


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Figure 11. Fluorescence microphotographs of DCFH-DA ROS detection Assay in EA.hy926 cells grown on A.Native collagen sheet, Collagen sheet cross-linked with B. PCNP (10µg/ml), C. PCNP (50µg/ml), D. PCNP (100µg/ml)

Figure 12. (a)- Phase contrast microphotographs of Tube Formation Assay in EA.hy926 cells grown on A.Native collagen sheet,Collagen sheet cross-linked with B. PCNPs (10µg/ml), C. PCNPs (50µg/ml), D. PCNPS (100µg/ml). (b)- Total Tube length of the endothelial tubes formed on Native as well as PCNP cross-linked collagen sheets


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Figure 13.Fluorescence microphotographs of Chicken Aortic arch Assay of EA.hy926 cells grown on Native collagen sheet, Collagen sheet cross-linked with PCNPs (10µg/ml), PCNPs (50µg/ml) and PCNPS (100µg/ml). Row a represents 0th day, b &c represents phase contrast and fluorescence images captured on 14th day

Figure 14.Total Tube length of the vascular sprouts formed on aortic arches placed on native as well as PCNP cross-linked collagen sheets ACS Paragon Plus Environment

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Figure 15.(a)- Phase contrast microphotographs of Stem cell Differentiation Assay in HWJMSC cells grown on A. Native collagen sheet, B. Collagen sheet cross-linked with PCNPs (10µg/ml), C. Collagen sheet cross-linked with PCNPs (50µg/ml), D. Collagen sheet cross-linked with PCNPS (100µg/ml) showing tube like morphology resembling Endothelial cells (b)- Total Tubule length of the differentiated HWJMSC cells placed on native as well as PCNP crosslinked collagen sheets


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Figure 16.Microphotographs of invivo wound healing assay carried out in male wistar rats. Row A- wound area applied with collagen sheets cross-linked with PCNP(10µg/ml) , Row B- wound area applied with collagen sheets cross-linked with PCNP(50µg/ml), Row C- wound area applied with collagen sheets cross-linked with PCNP(100µg/ml), Row D- wound area applied with Native collagen sheets. The images show accelerated healing of wounds in PCNP cross-linked collagen sheets. Wound images a,g,m,s represents 0th Day , b,h,n,t represents 3rd Day, c,i,o,u represents 7th Day, d,j,p,v represents 11th Day, e,k,q,w represents 15th Day and f,l,r,x represents 17th Day of various sample treated rats.


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Figure 17.Results of invivo wound healing assay in male wistar rats. Percentage reduction observed with collagen cross-linked with PCNPs of 10µg/ml, 50µg/ml and 100µg/ml compared to that of native collagen sheet and untreated saline control rats


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Figure 18.Microphotograph of Masson’s Trichrome Staining of 15th Day excised skin of male wistar rats treated with A. Native collagen, B. Saline control, C. Collagen+PCNP10, D. Collagen+PCNP50 and E. Collagen+PCNP100. Blood vessel and hair follicle are marked with Yellow and Red arrow marks respectively.


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TABLES Table 1.CD spectra wavelengths and Rpn Ratio of Native, Glutaraldehyde and PCNP crosslinked collagen sheets Wavelength (nm)

Rpn Ratio

Negative Peak Cross-over Positive peak Native collagen

197

214

221

0.115

Collagen+ Glutaraldehyde

198

214

221.5

0.101

Collagen+ PCNP(10µg/ml)

197.5

214

221

0.112


197.5

214

221

0.108


198

214

221.5

0.109

Table 2.TNBS Assay results of PCNP cross-linked collagen sheets compared to that of glutaraldehyde cross-linked and native collagen sheet Free Amino Group Present Native Collagen

Degree of Cross-linking

Percentage Cross-linking

0

0

Collagen+ Glutaraldehyde

0.30

0.70

70%


0.70

0.30

30%


0.60

0.40

40%


0.40

0.60

60%


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Table 3.Swelling ratio analysis’ results of PCNP cross-linked collagen sheets Percentage increase in swelling 12hr

Swelling Ratio

24hr

12hr

24hr

Native Collagen 30%

60%

0.3

0.6


70%

150%

0.7

1.5


45%

80%

0.45

0.8


20%

50%

0.2

0.5

Table 4. Hemocompatibility assay results showing good hemocompatibilty for PCNP crosslinked collagen sheets Hemolysis (%) Native Collagen

0.14


0.14


0.21


0.28


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