CONTROLLING STEM CELL DIFFERENTIATION FOR TISSUE ENGINEERING
SMITA PRABHAKAR PATIL
CENTRE FOR BIOMEDICAL ENGINEERING INDIAN INSTITUTE OF TECHNOLOGY DELHI
OCTOBER 2019
© Indian Institute of Technology Delhi (IITD), New Delhi, 2019
CONTROLLING STEM CELL DIFFERENTIATION FOR TISSUE ENGINEERING
by
SMITA PRABHAKAR PATIL Centre for Biomedical Engineering
Submitted
in fulfilment of the requirements of the degree of Doctor of Philosophy to the
INDIAN INSTITUTE OF TECHNOLOGY DELHI
OCTOBER 2019
Dedicated to Mum and Baba
CERTIFICATE
This is to certify that the thesis entitled ‘Controlling Stem Cell Differentiation for Tissue Engineering’ being submitted by Ms. Smita Prabhakar Patil to the Indian Institute of Technology Delhi for the award of degree of Doctor of Philosophy is a record of bonafide research work carried out by her. Ms. Smita Prabhakar Patil has worked under my guidance and supervision and has fulfilled the requirements for the submission of this thesis, which to our knowledge has reached the requisite standard.
The results contained in this thesis are original and have not been submitted, in part or full, to any other University or Institute for the award of any other degree or diploma.
Dr. Neetu Singh
Centre for Biomedical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016 India
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ACKNOWLEDGEMENTS
I would like to thank my PhD advisor, Dr. Neetu Singh, for her valuable guidance and continuous encouragement during all my PhD life. Her enthusiasm, passion towards research and her unique approach to solve problems has always inspired me to be a better researcher. Working under her supervision has been a truly enriching experience and I learnt and mastered many techniques in her laboratory. She also provided all the funding required to carry this research and gave me freedom to explore my research area. I am thankful to University Grants Commission, India for providing me research fellowship.
I would also like to express gratitude to SRC members, Prof. Harpal Singh, Prof. Veena koul, and Dr. Shalini Gupta, for their insightful suggestions and comments. I extend my sincere thanks to all the faculty and staff members of the Centre for Biomedical Engineering, for their guidance and generous support.
I would like to acknowledge all the past and current members of MRNBL, especially Sonal, Vartika, Anil, Navin, Sahil, Akshay, Dr. Tejinder, Dr. Ritu, Shreemoyee, Parul and Shweta for their love, help and moral support during all the up and downs of my PhD life. I am especially grateful to Sonal and Anil for interesting and insightful scientific discussions that not only helped me in troubleshooting research problems but also broadened my knowledge. I am thankful to Sahil, Shweta, Tejinder and Sirsendu for proofreading my thesis and articles.
I extend my sincere thanks to friends from IIT Delhi – Gopendra, Kritika, Thanusha, Sabeeha, Kirtika, Chetan, Dr. Aradhna, Dr. Arun for their support throughout my research. I am also grateful to Dr. Jasmin for her guidance in RT- PCR setup. I thank Dr. Dikshi and Neha for their help with confocal microscopy.
I gives me a great pleasure to thank my friends outside the IIT Delhi, especially Aditi, Veena, Rajesh, Aniket, Yogesh, Shraddha for their constant words of encouragement and cheering me up in my troubled times.
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I wish to thank my husband Sirsendu for all his support, patience and love.
Thank you for always listening to my rambling talks and for your understanding during this difficult phase of life.
I would also like to express my gratitude to my brother Akash, all the cousins and family members for their unending love and support throughout this journey. I am also indebted to my grandparents for their love and understanding.
I miss you Anna, thank you for all the words of motivation and your love, you will always remain in our hearts. Last but not the least I would like to thank my parents for their never ending support and understanding, without which this thesis would not have been possible. They never questioned me and gave me the freedom to live my life the way I wanted and their belief in me always motivates me to be a better person.
Smita Patil
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ABSTRACT
Human organs are extremely complex assemblies made by the collective, functional organization of multiple tissue types, in a spatially defined manner. Proper tissue function and regeneration rely on the spatial and temporal control of biophysical and biochemical cues, including soluble molecules, cell-cell contacts, cell-extracellular matrix contacts, and physical forces. To achieve controlled differentiation appropriate cues are required, if these cues are patterned on the surface in a desired manner, it can lead to a tissue or organ as the same happens during the developmental process in vivo. Consequently, from a basic point of view, the purpose of tissue engineering is fabrication of a biomaterial which can inherently provide a 3D microenvironment for cell growth, proliferation as well as guide formation of new tissue. Even though countless tissue engineering constructs have been developed, many of them are unable to completely mimic their native counterparts and are not as successful as anticipated during in vivo applications. Also, musculoskeletal degenerative diseases are the second largest reason for disability worldwide, thus this thesis focuses mainly on bone tissue engineering.
Lately, silk fibroin has been gaining a lot of attention as a scaffold for tissue engineering and many different types of cells such as stem cells, fibroblasts, osteoblasts, nerve cells have been grown and proliferated on silk fibroin based matrices. The high mechanical strength, biodegradability, and low inflammatory response of silk fibroin make it one of the favorable scaffolds for bone tissue engineering applications. Strategies used for modification of silk fibroin, to induce differentiation of human mesenchymal stem cells (hMSCs), include complex chemical modifications or incorporation of peptides in the fibroin itself. These strategies do not allow presentation of all functional groups on the surface and as the cell attachment is surface phenomenon, proper guidance of differentiation may not be achieved. A strategy for surface grafting of carboxylic acid groups via plasma induced graft polymerization method was developed recently. This strategy enables spatial control over cell adhesion on silk films, but spatially controlled differentiation of stem cells on silk films has not been investigated. Consequently, the surface modification of silk film with functional groups and its effect on the differentiation of stem cells without use of differentiation media, is probed in this thesis.
Chapter 1 discusses the challenges and recent developments in the field of tissue engineering. In chapter 2, silk fibroin surface has been modified by grafting functional
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groups. The simple strategy reported here, does not make use of any external supplements in the media for induction of differentiation and allows for spatial control over differentiation of hMSCs, by modulating functional groups on silk films. This study will foster the development of effective silk based tissue engineered constructs.
The ideal biomaterial for tissue engineering should not only be biocompatible, but also antibacterial; as implantation of a foreign material inside body increases chances of microbial infection. Most of the studies on the development of antibacterial materials do not investigate further into implications of the introduction of antimicrobials on stem cells differentiation. Therefore in chapter 3, development of antibacterial silk fibroin films containing in situ synthesized AgNPs by an easy, convenient and environment friendly means is investigated. Further, the antimicrobial efficacy of these films was evaluated against gram negative bacteria as well as antibiotic resistant bacteria and it was found to be effective against both. The cytocompatibility of these scaffolds was examined with fibroblast and osteoblast cells. Also the effect of AgNPs present in films, on osteogenic differentiation potential of hMSCs was studied and it was observed that the presence of AgNPs at lower concentrations did not have any detrimental effect.
Studying differentiation in 3 dimensions (3D), which mimics in vivo conditions is more relevant for clinical applications of tissue engineering. In chapter 4, synthesis of silk fibroin and alginate based beads is investigated for cell encapsulation and hMSCs differentiation. Use of alginate is advantageous as it is cheaper compared to other biopolymers and the encapsulated cells can be retrieved easily without causing undue stress to the cells. Proliferation and viability of osteoblast-like cells and hMSCs in these alginate- silk beads was also studied. As it has been demonstrated in chapter 2, that carboxylic groups help in chondrogenic differentiation and phosphate groups in osteogenic differentiation of hMSCs in 2D, here the effect of these groups in the 3D microenvironment on hMSCs proliferation and differentiation was evaluated. This study will help in understanding the hMSCs behavior in 3D as well as provide an easy to fabricate, simple and inexpensive in vitro model to study various aspects of hMSCs differentiation in the 3D system. Chapter 5 discusses the conclusion and future outlook of the thesis.
v साराांश
मानव के अवयव, एक स्थाननक रूप से परिभानित तिीके से, कई ऊतक प्रकाि ों के सामूनिक, कायाात्मक सोंगठन द्वािा बनाए गए अत्योंत जनिल सोंय जन िैं। उनित ऊतक काया औि पुनरुत्पादन, जैवभौनतक औि जैव
िासायननक सोंकेत ों के स्थाननक औि लौनकक ननयोंत्रण पि ननभाि किते िैं, नजसमें समाधेय व्यूिाणु, क निका- क निका सोंपका, क निका -बाह्य मैनििक्स सोंपका औि भौनतक बल िानमल िैं। ननयोंनत्रत नवभेदीकिण क प्राप्त किने के नलए उपयुक्त सोंकेत ों की आवश्यकता ि ती िै, यनद ये सोंकेत सति पि वाोंनित तिीके से लगाए जाते
िैं, त यि ऊतक या अवयव क जन्म दे सकता िै जैसा नक एक जीनवत जीव में नवकास प्रनिया के दौिान
ि ता िै। नतीजतन, एक मूल दृनिक ण से, ऊतक इोंजीननयरिोंग का उद्देश्य एक जैव-पदाथा का ननमााण िै ज क निका वृद्धि, प्रसाि के साथ-साथ नए ऊतक के मागादिाक गठन के नलए स्वाभानवक रूप से एक 3 डी
सूक्ष्म पयााविण प्रदान कि सकता िै। भले िी अननगनत ऊतक इोंजीननयरिोंग ननमााण नवकनसत नकए गए िैं, उनमें से कई अपने मूल समकक् ों की पूिी तिि से नकल किने में असमथा िैं औि जीनवत जीव में अनुप्रय ग ों
के दौिान अपेनक्त रूप से सफल निीों थे। इसके अलावा, मस्कुल स्केलेिल अपक्यी ि ग दुननया भि में
नवकलाोंगता का दूसिा सबसे बडा कािण िै, नलिाजा यि ि ध प्रबोंध मुख्य रूप से िड्डी ऊतक इोंजीननयरिोंग पि केंनित िै।
िाल िी में, िेिम फाइब्र इन, ऊतक इोंजीननयरिोंग के नलए एक स्काफ ल्ड के रूप में बहुत ध्यान आकनिात कि ििा िै औि कई अलग-अलग प्रकाि की क निकाएों जैसे मूल क निका, तोंतुप्रसू, अद्धस्थक िक, तोंनत्रका क निका िेिम फाइब्र इन आधारित मैनििसेस पि उगाए औि प्रिुि द्भवन नकये गए िै। िेिम फाइब्र इन की उच्च याोंनत्रक िद्धक्त, जैनवक पतन क्माता औि कम सूजन प्रनतनिया इसे अद्धस्थ ऊतक इोंजीननयरिोंग अनुप्रय ग ों के नलए अनुकूल स्काफ ल्ड में से एक बनाती िै। िेिम फाइब्र इन के सोंि धन के नलए उपय ग की
जाने वाली िणनीनतयााँ, मानव मध्य तक मूल क निकाओों (hMSC) के नवभेदन क प्रेरित किने के नलए, जनिल
िासायननक परिवतान या फाइब्र इन में पेप्टाइड्स क सद्धिनलत किना िानमल िैं। ये िणनीनतयााँ सति पि सभी
कायाात्मक समूि ों की प्रस्तुनत की अनुमनत निीों देती िैं औि जैसा नक क निका सोंलगन सति की घिना िै, नवनििीकिण का उनित मागादिान िायद िानसल निीों नकया जा सकता िै। प्लाज्मा प्रेरित ग्राफ्ट बहुलीकिण नवनध के माध्यम से काबोद्धिनलक एनसड समूि ों की सति ग्राद्धफ्टोंग के नलए एक िणनीनत िाल िी में नवकनसत की गई थी। यि िणनीनत िेिम नफल् ों पि क निका आसोंजन पि स्थाननक ननयोंत्रण क सोंभव बनाती िै, लेनकन
िेिम नफल् ों पि मूल क निकाओों के स्थाननक रूप से ननयोंनत्रत नवनििीकिण की जाोंि निीों की गई िै।
नतीजतन, कायाात्मक समूि ों के साथ िेिम नफल् की सति का परिवतान औि भेदभाव मीनडया के उपय ग के नबना मूल क निकाओों के नवनििीकिण पि इसका प्रभाव इस ि ध प्रबोंध में जाोंि की इस ि ध प्रबोंध में
जाोंि की गई िै जाती िै।
अध्याय 1 ऊतक इोंजीननयरिोंग के क्ेत्र में िुनौनतय ों औि िाल के घिनािम ों पि ििाा किता िै। अध्याय 2 में, िेिम फाइब्र इन सति क कायाात्मक समूि ों क ग्राफ्ट किके सोंि नधत नकया गया िै। यिाों बताई गई
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सिल िणनीनत, नवभेदीकिण क िानमल किने के नलए मीनडया में नकसी भी बाििी पूिक का उपय ग निीों
किती िै औि िेिम नफल् ों पि कायाात्मक समूि ों क सोंि नधत किके hMSC के नवनििीकिण पि स्थाननक ननयोंत्रण की अनुमनत देती िै। यि अध्ययन प्रभावी िेिम आधारित ऊतक इोंजीननयरिोंग ननमााण ों के नवकास क बढावा देगा। ऊतक इोंजीननयरिोंग के नलए आदिा जैव-पदाथा न केवल जैव-सोंगत ि ना िानिए, बद्धि
जीवाणुि धी भी ि ना िानिए; क् ोंनक ििीि के अोंदि एक नवदेिी सामग्री के आि पण से जीवाणु सोंिमण की
सोंभावना बढ जाती िै। जीवाणुि धी इोंप्लाोंि सामग्री के नवकास पि अनधकाोंि अध्ययन, मूल क निका
नवभेदीकिण पि प्रनतसूक्ष्मजीवाणुक की समावेिन के प्रभाव की आगे जाोंि निीों किते िैं। इसनलए अध्याय 3 में, जीवाणुि धी िेिम फाइब्र इन नफल् ों के नवकास में एक आसान, सुनवधाजनक औि पयााविण के अनुकूल साधन ों द्वािा AgNP क सोंश्लेनित नकया गया िै। इसके अलावा, इन नफल् ों की ि गाणुि धी प्रभावकारिता का
मूल्ाोंकन ग्राम नेगेनिव बैक्टीरिया के साथ-साथ एोंिीबाय निक प्रनति धी बैक्टीरिया के द्धिलाफ नकया गया था
औि यि द न ों के द्धिलाफ प्रभावी पाया गया था। इन स्काफ ल्ड की जैव-सोंगती की जाोंि तोंतुप्रसू औि
अद्धस्थक िक क निकाओों के साथ की गई थी। इसके अलावा, hMSCs की ओस्ट जेननक नवनििीकिण क्मता
पि नफल् ों में मौजूद AgNP के प्रभाव का अध्ययन नकया गया औि यि देिा गया नक, कम साोंिता में AgNP की उपद्धस्थनत का क ई िाननकािक प्रभाव निीों पडा।
3 आयाम ों (3D) में नवनििीकिण का अध्ययन किना, ज नक इन नविि परिद्धस्थनतय ों की नकल किता
िै, ऊतक इोंजीननयरिोंग के क्लीननकल अनुप्रय ग ों के नलए अनधक प्रासोंनगक िै। अध्याय 4 में, िेिम फाइब्र इन औि एद्धिनेि आधारित दान ों के सोंश्लेिण क क निका सोंपुिीकिण औि hMSC नवनििीकिण के नलए जाोंि
की गई िै। एद्धिनेि का उपय ग लाभप्रद िै कािण अन्य बाय पॉनलमि ों की तुलना में एद्धिनेि सस्ता िै औि
क निकाओों पि अनुनित तनाव पैदा नकए नबना सोंपुनित क निकाओों क आसानी से पुनः प्राप्त नकया जा
सकता िै। इन एद्धिनेि-नसि दान ों में अद्धस्थक िक जैसी क निकाओों औि hMSCs के प्रसाि औि
व्यविायाता का भी अध्ययन नकया गया िै। जैसा नक अध्याय 2 में प्रदनिात नकया गया िै, नक काबोद्धिनलक समूि 2D में hMSC के िॉन्ड्ि जेननक नवनििीकिण औि फॉस्फेि समूि ओद्धस्टय जेननक नवनििीकिण में मदद किते िैं, यिााँ hMSCs प्रसाि औि नवनििीकिण पि 3D माइि एन्वायिमेंि में इन समूि ों के प्रभाव का
मूल्ाोंकन नकया गया िै। यि अध्ययन 3D में hMSC के व्यविाि क समझने में मदद किेगा औि साथ िी 3D नसस्टम में hMSC नवनििीकिण के नवनभन्न पिलुओों का अध्ययन किने के नलए इन नविि मॉडल में एक आसान, सिल औि सस्ता ननमााण प्रदान किेगा। अध्याय 5 में ि ध प्रबोंध के ननष्किा औि भावी दृनिक ण पि ििाा की
गई िै।
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Table of Contents
Acknowledgements i
Abstract iii
List of Figures xiii
List of Tables xxi
List of Schemes xxiii
List of Abbreviations xxv
Chapter 1: Introduction and Literature Review
1.1 Motivation and Background……… 3
1.2 Key Challenges in the Field………. 4
1.3 Tissue Engineering………... 6
1.3.1 Cells in Tissue Engineering………. 7
1.3.2 Scaffold for Tissue Engineering……….. 9
1.3.2.1 Ceramics………... 9
1.3.2.2 Natural Biomaterials………. 10
1.3.2.3 Synthetic Polymers………... 12
1.3.3 Directing Stem Cell Differentiation for Tissue Engineering………...… 12
1.3.3.1 Surface Chemistry……… 13
1.3.3.2 Growth Factors………. 13
1.3.3.3 Modulus of a Material……….. 16
1.3.3.4 Surface Topography………. 16
1.4 Silk Fibroin as a Tissue Engineering Scaffold………. 17
1.5 Antibacterial Materials for Bone Tissue Engineering………. 20
1.6 3D Silk Scaffold for hMSCs Differentiation ……….. 22
1.7 Objectives of the Study……… 24
1.8 Outline of the Thesis……… 24
References……….. 26
viii
Chapter 2: Spatially Controlled Functional Group Grafting of Silk Films to Induce Differentiation of hMSCs
2.1 Introduction….. ……….. 43
2.2 Materials and Methods……… 46
2.2.1 Materials……….. 46
2.2.2 Preparation of an Aqueous Solution of Silk Fibroin and Silk Films…… 47
2.2.3 Functionalization of the Silk Films by Graft Polymerization….………. 47
2.2.4 Contact-Angle Measurements for Monitoring Changes in the Hydrophilicity………. 48
2.2.5 Carboxyl Group Determination…...……… 48
2.2.6 Phosphate Group Characterization by Pro Q Diamond Phosphoprotein Staining.………... 48
2.2.7 Cell Culture……….. 49
2.2.7.1 Cell Viability Assay………. 49
2.2.7.2 MTT Assay………... 49
2.2.8 Cultivation of hMSCs…… ………. 50
2.2.8.1 Cellular behavior of hMSCs on Silk Films………. 50
2.2.8.2 Morphological changes in hMSCs……….. 50
2.2.8.3 Calcium Assay………. 51
2.2.8.4 Immunocytochemical Staining……… 51
2.2.8.5 mRNA Isolation, cDNA Synthesis and Gene Amplification using Real Time Polymerase Chain Reaction (PCR)………... 51
2.2.9 Preparation of Hybrid SF………. 52
2.2.10 Alizarin Red S Staining ……… 53
2.2.11 Statistical Analysis………. 53
2.3 Results ………. 53
2.3.1 Silk Film Functionalization………. 53
2.3.2 Cell Adhesion and Proliferation on Silk Films……… 55
2.3.3 Studies with hMSCs………. 57
2.3.3.1 Viability and Proliferation of hMSCs………... 57
2.3.3.2 Morphological Changes in hMSCs………... 59
2.3.3.3 Calcium Content of Cells………. 59
ix
2.3.3.4 Differentiation of hMSCs……….. 60
2.4 Discussion ………... 63
2.5 Conclusion………... 65
References……….. 67
Chapter 3: Development of Antibacterial Silk Fibroin Scaffolds with Green Synthesized Silver Nanoparticles for Bone Tissue Engineering Applications
3.1 Introduction….. ………... 733.2 Materials and Methods………. 76
3.2.1 Materials……….. 76
3.2.2 Aqueous Silk Fibroin Solution Preparation………. 76
3.2.3 In-situ synthesis of Silver Nanoparticles in Silk Fibroin Films…….….. 76
3.2.4 Particle Characterization………..……… 77
3.2.5 Release Studies…...……….……… 78
3.2.6 Antimicrobial Activity of Silk Films with AgNPs.……….…….……… 78
3.2.6.1 Planktonic Bacteria Assessment………... 78
3.2.6.2 Activity of Sessile Bacteria……….. 78
3.2.6.3 Colony Count Method……….………. 79
3.2.6.4 Determination of MIC……….………. 79
3.2.7 In vitro Cytocompatibility and Osteogenic Activity Studies………. 80
3.2.7.1 Cell Culture……….……….………… 80
3.2.7.2 Alamar Blue Assay……….………. 80
3.2.7.3 Apoptosis Assay………... 80
3.2.7.4 Intracellular ROS Assay……….. 81
3.2.7.5 Live Dead Staining Assay………...…... 81
3.2.8 In vitro Bacterial Infection Model………...…… 81
3.2.9 Calcium Assay……….……… 82
3.2.10 Osteogenic Differentiation of hMSCs……… 82
3.2.11 Statistical analysis……….. 82
3.3 Results……….………. 82
x
3.3.1 In situ Synthesis of Silver Nanoparticles and Silk Fibroin Film
Formation……….. 82
3.3.2 Characterization of AgNPs……… 83
3.3.3 Antimicrobial Activity of Silk Films with AgNPs………. 89
3.3.4 In vitro Cytocompatibility of Films………... 90
3.3.5 In vitro Bacterial Infection Model………... 94
3.3.6 Calcium Assay………... 95
3.3.7 Osteogenic Differentiation of hMSCs……… 96
3.4 Discussion ………. 97
3.5 Conclusion………. 100
References……… 102
Chapter 4: Effect of functional groups on human mesenchymal stem cell differentiation in 3D beads
4.1 Introduction….. ……….. 1094.2 Materials and Methods……… 112
4.2.1 Materials……….. 112
4.2.2 Alginate Phosphorylation ………... 113
4.2.3 Cell Culture…….……….………. 113
4.2.3.1 Cultivation of hMSCs ……… 113
4.2.4 Bead Fabrication …...……… 114
4.2.5 Coomassie Brilliant Blue (CBB) Staining and BCA Assay………… 114
4.2.6 Live Dead Staining Assay……….……….……….……… 115
4.2.7 Scanning Electron Microscopy (SEM) ……….. 115
4.2.8 Cell Proliferation Study ………. 115
4.2.9 Immune Response Studies ………. 116
4.2.10 Calcium Assay ……….… 116
4.2.11 Immunocytochemical Staining ……….…….... 116
4.2.12 mRNA Isolation, cDNA Synthesis and Gene Amplification using Real Time Polymerase Chain Reaction (qPCR).…………..……….. 117
4.2.13 Alizarin Red S and Safranin O Staining ……….. 117
xi
4.2.14 Statistical Analysis………...………. 117
4.3 Results ………...………. 118
4.3.1 Alginate Phosphorylation……… 118
4.3.2 Bead Fabrication and Characterization……… 119
4.3.3 Cell Viability and Proliferation of Encapsulated Cells………... 123
4.3.4 Immune Response Studies………... 124
4.3.5 Calcium Content of Cells……… 125
4.3.6 Differentiation of hMSCs...………. 125
4.4 Discussion ……….. 129
4.5 Conclusion……….. 131
References………. 133
Chapter 5: Conclusions and Future Outlook
5.1 Conclusions….. ……….. 1395.2 Future Outlook………. 142
Publications 143
Curriculum vitae 145
xiii
List of Figures
Figure
Number Title Page
No.
Chapter 1
Figure 1.1. Processing of aqueous silk solution into various morphologies. 17
Chapter 2
Figure 2.1. Increase in carboxylic acid residues (~2 times) after grafting of poly acrylic acid (pAAc) on silk film (SF). The amount of carboxylic acid grafted was determined using toluidine blue o assay.
53
Figure 2.2. Characterization of carboxylic acid and phosphate group grafting (A) water contact angle measurements and (B) Pro Q Diamond staining. Due to presence of carboxylic acid groups, pAAc-SF showed decrease in contact angle compared to bare silk film (SF).
Decrease in the contact angle and increase in the fluorescence intensity confirms phosphate grafting on silk films. (pAAc-SF:
polyacrylic acid grafted SF, PE-SF: o-phoshphoethanolamine grafted SF) (One way ANOVA, *** p<0.001).
54
Figure 2.3. Attenuated total reflection-fourier transform infrared (ATR-FTIR) spectroscopy confirming presence of β sheet formation in silk film (SF), poly acrylic acid grafted (pAAc-SF) and phosphate grafted (PE-SF) silk films.
54
Figure 2.4. Surface morphology of films. SEM images of silk film (SF), poly acrylic acid grafted (pAAc-SF) and phosphate grafted (PE-SF) silk films showing no change in morphology due to grafting of
functional groups.
55
Figure 2.5. Cell viability assay using Calcein AM (A), (C) and quantification of cell adhesion by MTT (B), (D) with Hela, NIH 3T3 cells, respectively. (E) Calcein AM staining and (F) MTT assay for hMSCs cultured onto SF, pAAC-SF and PE-SF for 24 h while (G)
and (H) are for 72 h culture time. Green: Live cells stained with 56
xiv
calcein AM, Scale bar: 100 μm. (SF: silk film, pAAc: polyacrylic acid grafted SF, PE: o-phoshphoethanolamine grafted SF) (One way ANOVA, *** p<0.001, * p<0.05).
Figure 2.6. Cell adhesion and proliferation of hMSCs. (A) Time dependent cell adhesion of hMSCs onto Tissue Culture Polystyrene (TCP), silk film (SF), polyacrylic acid grafted (pAAc-SF) and phosphate grafted (PE-SF) films, showing faster adhesion at 0.5 h on acrylic acid and phosphate grafted films. (B) Proliferation of hMSCs was also studied using alamar blue, at different time intervals upto 12 days after seeding the cells onto the silk films. Increase in fluorescence intensity indicates increased cell density. (One way ANOVA, *** p<0.001, ** p<0.01, * p<0.05, the p values indicate the significant increase with respect to 0 h/ day 0 values of the same sample)
57
Figure 2.7. Morphological studies. SEM images of hMSCs grown on SF, pAAc-SF and PE-SF for three and 60 days showing change in morphology and proliferation on the films. Black arrows indicate cells with distinguished morphology. (SF: fibroblast like, pAAc-SF:
chondrocyte like, PE-SF: osteocyte like)
58
Figure 2.8. Calcium content determined after culturing hMSCs on films and control for 60 days. The values were normalized with cell number extrapolated by alamar blue assay. SF: silk film, pAAc-SF:
polyacrylic acid grafted SF, PE-SF: o-phoshphoethanolamine grafted SF, TCP: Tissue culture polystyrene control. (One way ANOVA, *** p<0.001, ** p<0.01.)
58
Figure 2.9. Immunostaining of hMSCs grown on silk films for 60 days and stained with antibodies against osteocalcin (A) and collagen II (B).
The secondary antibody used in this assay was Alexa Fluor 488 tagged. Nuclei are stained with DAPI. (SF: silk film, pAAc:
polyacrylic acid grafted SF, PE: o-phoshphoethanolamine grafted SF). Blue: DAPI, Green: Alexa Fluor 488. Objective used for imaging: 20x; Scale bar: 50 μm.
59
Figure 2.10. Gene expression analysis of (A) RUNX2, (B) OCN and (C) COL2A1 genes, after 60 days of hMSC culture on films using real time PCR. Relative fold change in each gene expression level compared to control was calculated by ΔΔCt method by normalizing the values with GAPDH as internal control. (One way ANOVA, ***
p<0.001, ** p<0.01).
60
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Figure 2.11. Positive controls for osteogenic markers RUNX2 and OCN. Gene expression analysis of genes using real time PCR was carried out after 21 days, for hMSC cultured on silk films in presence of 100 μg/mL of bone morphogenic protein 2 (BMP 2). Relative fold change in each gene expression level compared to control (hMSCs grown on tissue culture polystyrene without BMP) was calculated by ΔΔCt method by normalizing the values with GAPDH as internal control.
61
Figure 2.12. Alizarin red S staining of hMSCs grown for 60 days on hybrid silk film containing regions grafted with carboxylic acid (pAAc) and phosphate (PE). hMSCs present at interface (A), in carboxylic acid grafted regions (B) and phosphate grafted region (C,D) are imaged using bright field microscopy. Nodule formation (red arrow) a characteristic of osteocytes can be seen in figure A and C, nodules had more stain. White arrows indicate cell layers which are peeling off. Scale bar:100µm.
61
Figure 2.13. Immunostaining of hMSCs grown for 60 days, on hybrid silk film with half surface carboxylic acid and other half with phosphate grafted regions and stained with antibody against collagen II and osteocalcin. The secondary antibody used in this assay was Alexa Fluor 594 for collagen II and Alexa Fluor 488 for osteocalcein.
Nuclei are stained with DAPI. White arrows indicate staining with osteocalcin. Blue: DAPI, Green: Alexa Fluor 488, Red: Alexa Fluor 594. Objective used for imaging: 20×.
62
Figure 2.14. Immunostaining of hMSCs grown for 60 days, on hybrid silk film with half surface carboxylic acid and other half with phosphate grafted regions and stained with antibody against collagen II and osteocalcin. The phosphate grafted region stained negative for collagen and acrylic acid grafted region was negative for osteocalcein. The secondary antibody used in this assay was Alexa Fluor 594 for collagen II and Alexa Fluor 488 for osteocalcein.
Nuclei are stained with DAPI. White arrows indicate staining with osteocalcin. Blue: DAPI, Green: Alexa Fluor 488, Red: Alexa Fluor 594. Objective used for imaging: 20×.
63
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Chapter 3
Figure 3.1. In situ synthesis of AgNPs using silk fibroin solution and formation of silk films. (A) Color change over time due to light exposure in silk fibroin solution with varying AgNO3 concentrations. (B) Dried silk fibroin films held with forceps.
83
Figure 3.2. UV-visible spectra of silk fibroin solution alone and with different AgNO3 concentration over a 50 h time period with continuous white
light exposure, confirming formation of AgNPs. 84 Figure 3.3. UV-visible spectra of silk fibroin solution alone and with varying
AgNO3 concentrations after 30 h white light exposure, showing
absorption peak at 405-425 nm, confirming formation of AgNPs. 85 Figure 3.4. Attenuated total reflection-fourier transform infrared (ATR-FTIR)
spectroscopy confirming β sheet formation even after in situ synthesis of AgNPs in silk film.
85
Figure 3.5. Scanning electron micrograph of only silk film (SF) and silk film with AgNPs using 0.5% AgNO3 (w/w to silk fibroin).
86
Figure 3.6. Water contact angle measurements of silk films with varying concentrations of AgNO3 used for AgNP synthesis.
86
Figure 3.7. (A) Transmission electron micrograph of AgNPs synthesized using different AgNO3 concentrations (B) Percent frequency distribution of AgNP diameter sizes (C) Elemental analysis by energy dispersive X ray showing peak signals specific to silver confirming presence of AgNPs.
87
Figure 3.8. (A) TGA analysis of silk films showing decrease in weight, first due to loss of water and then degradation of silk fibroin. (B) Amount of Ag released from SF with 0.75 % AgNO3 in presence of PBS and protease.
88
Figure 3.9. Antibacterial activity of AgNP containing silk films on (A) planktonic S. aureus and (B) biofilm formation of S. aureus after 24 h incubation using bacteria grown on tissue culture plate (TCP) as a control. The effect of antibiotics was studied against bacteria grown on SF.
89
xvii
Figure 3.10. MIC determination by microbroth dilution method. (A) Ampicillin and kanamycin show MIC of 8 μg/mL for both bacteria. (B) AgNPs of 20 nm diameter show MIC of 20 nM.
90
Figure 3.11. Antibacterial activity of AgNP containing silk films against E. coli resistant to (A) Kanamycin (B) Ampicillin antibiotic by colony count method. Bacterial suspension added in an empty well was used as control. (***p<0.001)
91
Figure 3.12. Antimicrobial activity evaluation by colony count method. Images of agar plates showing growth of colonies after overnight incubation confirming antibacterial activity of AgNP containing silk films against biofilm forming antibiotic resistant bacteria.
91
Figure 3.13. Influence of AgNO3 concentration on proliferation of (A) NIH3T3 fibroblast cells and (B) MG-63 osteoblast cells by alamar blue assay.
92
Figure 3.14. Flow cytometry analysis of MG-63 cells grown on silk films for (A) 24 h and (B) 4 days, showing increase in AgNP concentration caused increased apoptosis. After 4 day incubation, the positive control (TCP) showed 80 % viable cells while SFs with 0.5 % AgNO3 had 59 % cells which were viable. Apoptotic: early and late apoptotic cells. Positive control: Cells grown directly on tissue culture polystyrene plate.
92
Figure 3.15. Fluorescence microscopy images of live dead assay of fibroblast cells (NIH3T3) and osteoblasts (MG-63) grown on silk film with AgNPs for 24 h. Objective used: 20x, Merged images-Green: Live cells, Red: Dead cells and Bright field.
93
Figure 3.16. Intracellular ROS assay using MG-63 cells, showing generation of ROS due to AgNPs present in the scaffolds. Positive control: Cells grown on SF and incubated with H2O2 for 20 mins before addition of H2DCFDA (Scale bar: 50µm).
93
Figure 3.17. Fluorescence microscopy images of calcein AM stained MG-63 cells post 24 h infection with S. aureus bacteria showing live cells (green).
94
Figure 3.18. Antibacterial activity of AgNP containing films seeded with MG-63 cells against biofilm forming S. aureus bacteria by colony count method.
95
Figure 3.19. Colony count experiment. Images of agar plates showing growth of colonies after overnight incubation confirming antibacterial activity
xviii
of AgNP containing silk films against biofilm forming S. aureus
bacteria. 95
Figure 3.20. Calcium assay of MG-63 cells grown on silk films (with varying concentrations of AgNPs) for 1 and 7 days showing the films help cells to maintain their characteristics.
96
Figure 3.21. Colony count experiment of films incubated with PBS for 7 days.
(A) Images of agar plates showing growth of colonies after overnight incubation confirming antibacterial activity of AgNP containing silk films against S. aureus bacteria is retained even after 7 days of PBS incubation. (B) Percent growth of bacteria compared to positive control.
96
Figure 3.22. Real time PCR study: Gene expression analysis of osteogenic markers viz. osteocalcin (OCN) and RUNX2 of hMSCs after 21 day incubation with BMP-2 (*** p<0.001, ** p<0.01, * p<0.05).
97
Chapter 4
Figure 4.1. 31P-NMR spectra of o-phosphoethanolamine and alginate conjugated with o-phosphoethanolamine (Alg-PO4).
118
Figure 4.2. Gels stained with Coomassie Brilliant Blue R-250, the blue color in gels confirms presence of the silk fibroin protein. The image was captured and then pseudo coloured using UVP’s IT2 gel documentation system.
119
Figure 4.3. Scanning electron micrograph of lyophilized beads showing their porous nature.
119
Figure 4.4. Degradation study of beads (A) Concentration of silk fibroin protein determined by BCA assay showing degradation of silk fibroin in presence of protease. (B) Beads stained with Coomassie Brilliant Blue R-250, the blue color in gels confirms presence of the silk fibroin protein. The image was captured and then pseudo coloured using UVP’s IT2 gel documentation system. (Two way ANOVA,
*** p<0.001)
120
Figure 4.5. Attenuated total reflection-fourier transform infrared (ATR-FTIR) spectroscopy confirming presence of β sheet formation in beads containing silk fibroin after 28 days of incubation in HBSS.
120
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Figure 4.6. (A) Scanning electron micrograph of ethanol treated Alg-SF gels (B) Scanning electron micrograph of MG-63 cells loaded onto ethanol treated Alg-SF gels, after 14 days of incubation showing presence of cells only on surface.
121
Figure 4.7. Live dead staining of alginate-silk fibroin bead treated with ethanol and then seeded with MG-63 cells after 14 days of incubation showing (A) fluorescence microscopy image (B) bright field image.
Green: viable cells. Scale bar: 100 µm.
121
Figure 4.8. Cytocompatibility of the scaffolds on MG-63 and hMSCs (A) Fluorescence microscopy images of live dead staining of MG-63 cells seeded beads after 24 h and 72 h incubation (merged image:
red, green, Green: viable cells, Red: dead cells). Scale bar: 100 µm.
122
Figure 4.9. Effect of initial cell seeding density on proliferation of cells seeded in beads after 24 h incubation, after normalization, using alamar blue assay.
122
Figure 4.10. Effect of composition on MG-63 cell proliferation in beads, using cell seeding density as (A) 0.5x105 cells/mL and (B) 105 cells/mL.
123
Figure 4.11. Proliferation of hMSCs in beads using alamar blue assay. 123 Figure 4.12. Fluorescence microscopy images of live dead staining of hMSCs
seeded beads after 24h and 72h incubation (merged image: red, green).
124
Figure 4.13. Scanning electron micrograph of lyophilized beads, 7 days after hMSCs seeding.
124
Figure 4.14. Study of the immune response of differentiated THP1 macrophages against only gels and hMSCs seeded gels by ELISA for detection of (A) IL-6 and (B) TNF α, showing that the beads are non- immunogenic.
125
Figure 4.15. Calcium content of hMSCs cultured on tissue culture plate (TCPS) and in beads after 28 days of culturing in complete media/
osteogenic differentiation media. (Two-way ANOVA, *** p<0.001,
** p<0.01)
126
Figure 4.16. Confocal microscopy images of hMSCs in gels after 14 days of incubation in cell culture media. The hMSCs were stained with
antibodies against collagen II (COL2) and osteocalcin (OCN). Blue: 126
xx
DAPI, Red: Alexa Fluor 594, Green: Alexa Fluor 488. Objectives used for imaging: 20x, 60x. Scale bar:50 µm.
Figure 4.17. Confocal microscopy images of hMSCs in gels after 28 days of incubation in basal media or differentiation media. The hMSCs were stained with antibodies against collagen II (COL2) and osteocalcin (OCN). Blue: DAPI, Red: Alexa Fluor 594, Green: Alexa Fluor 488.
Objectives used for imaging: 20x, 60x. Scale bar:50 µm.
127
Figure 4.18. Gene expression study by real time PCR to determine the effect of functional groups in the beads on hMSCs’ differentiation after 14 days incubation.
128
Figure 4.19. Staining of hMSCs after 28 days of incubation with gels, with (A) alizarin red s confirming presence of calcium depositions and (B) alcian blue confirming presence of glycosaminoglycans. Scale bar:100 µm.
128
xxi
List of Tables
TableNumber Table Title Page
Number Chapter 1
Table 1.1. Studies on effects of various surface chemistries on stem cell differentiation.
14 Table 1.2. Studies on the use of various peptide sequences and growth
factors that direct stem cell differentiation.
15 Table 1.3 Various tissue engineering applications of silk fibroin
scaffolds.
18
Chapter 2
Table 2.1 Primer sequences used for real time PCR. 52
xxiii
List of Schemes
SchemeNumber Scheme Title Page
Number Chapter 2
Scheme 2.1. Schematic representation of functional group grafted hybrid silk film and resulting hMSCs differentiation
65
Chapter 3
Scheme 3.1. Schematic of in situ AgNP synthesis and assessment of its antibacterial activity on films seeded with MG-63 cells (osteoblasts).
94
Chapter 4
Scheme 4.1. Fabrication of 3D beads for hMSCs differentiation. 129
xxv
List of Abbreviations
°C Degrees Celsius
µg Microgram
µL Microliter
2D 2 Dimentional
3D 3 Dimentional
AgNP Silver nanoparticles
AgNP-SFs AgNPs containing silk films
Ala Alanine
Alg Alginate
Alg-PO4 Phosphorylated alginate AuNP Gold nanoparticles
BMP Bone morphogenetic protein BSA Bovine serum albumin Calcein AM Calcein acetoxymethyl ester CBB Coomassie Brilliant Blue cDNA Complementary DNA CFU Colony forming units
COL2A1 Collagen type II alpha 1 chain D2O Deuterium Oxide
DAPI 4',6-diamidino-2-phenylindole
DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic acid ECM Extra-cellular matrix
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay ESC Embryonic stem cells
FACS Fluorescence-activated cell sorting FBS Fetal bovine serum
FDA Food and Drug Administration (USA) FGF-2 Fibroblast growth factor 2
xxvi
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
Gly Glycine
H2DCFDA 2',7'-dichlorodihydrofluorescein diacetate HA Hyaluronic acid
HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid hMSC Human mesenchymal stem cells
IL-6 Interleukin -6 kDa Kilodalton
LB broth Luria Bertani broth LSCs Limbal stem cells
MES 4-Morpholineethanesulfonic acid
mg Milligram
MIC Minimum inhibitory concentration
mL Mililiter
mM Millimolar
mm Millimeter
MRSA Methicillin-resistant Staphylococcus aureus MSC Mesenchymal stem cells
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MWCO Molecular Weight Cut Off
NHS N-Hydroxysuccinimide
nM Nanomolar
NMR Nuclear Magnetic Resonance NP Nanoparticles
NSCs Neuronal stem cells OCN Osteocalcin
pAAc Poly acrylic acid
pAAc-SF poly acrylic acid grafted silk films PBS Phosphate Buffered Saline
PDGF Platelet-derived growth factor PEG Poly ethylene glycol
PE-SF Phosphate grafted silk films PFA Paraformaldehyde
xxvii PGA Polyglycolic acid
PI Propidium iodide PLA Poly-l-lactic acid
PLGA Poly-dl-lactic-co-glycolic acid RNA Ribonucleic acid
rpm Rotations per minute
RT-PCR Real Time Polymerase Chain Reaction RUNX2 Runt related transcription factor 2
Ser Serine
SF Silk fibroin
TCP Tissue culture polystyrene plates TEM Transmission Electron Microscopy TGA Thermo gravimetric analysis
Thr Threonine
TNF α Tumor necrosis factor alpha USSCs Unrestricted somatic stem cells UV Ultraviolet
Val Valine
VEGF Vascular endothelial growth factor VIS Visible Spectroscopy
