CHAPTER 1 GENERAL INTRODUCTION

Transcription

1 1 CHAPTER 1 GENERAL INTRODUCTION 1.1 INTRODUCTION The loss or failure of an organ or tissue caused by trauma or disease is one of the most frequent and devastating problems in healthcare. Organ transplantation is the first choice to reconstruct the devastated tissues or organs. However, these surgical treatments have been facing certain challenges such as shortage of donated organs and immune rejection. Tissue engineering has emerged as an alternative approach to the conventional organ and tissue transplantation (Lalan et al 2001). Tissue engineering is a multidisciplinary field aiming to develop biological substitutes to restore, replace or regenerate defective tissues. Cells, scaffolds and growthstimulating signals are generally referred as the key components of engineered tissues. Scaffolds, typically made of polymeric materials, provide the structural support for cell attachment and subsequent tissue development (Langer & Vacanti 1993). Attempts have been made to fabricate scaffolds to mimic the chemical composition and structural properties of the extracellular matrix (ECM) because a tissue engineered scaffold with these characteristics will have a better chance at enhancing tissue regeneration in the body. Nanofibers are attractive scaffolds for tissue regeneration applications because they structurally mimic the native ECM (Wang et al 2013).

2 2 1.2 NANOFIBROUS SCAFFOLDS Electrospinning has been recognized as one of the most proficient technique to fabricate polymeric nanofibrous scaffolds. Various synthetic polymers such as poly ( -caprolactone) (PCL), poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (lactic-co-glycolic acid) (PLGA), polystyrene, polyurethane (PU), polyethyelene terephthalate (PET) and poly (L-lactic acidco-caprolactone) (PLACL) have been electrospun (Jie et al 2010). The diameter of the electrospun polymer ranges from micrometers to nanometers there by providing large surface area and high porosity that are important from tissue engineering point of view (Agarwal et al 2008). Nanofibers can also be electrospun into various patterns depending on the applications such as aligned, random, spiral, tubular, and sheath membrane. Every pattern has its own advantage, of which random nanofibers are generally used as scaffold in tissue engineering. The additional advantage of electrospinning is that the blended nanofibers can also be prepared by mixing polymer with other materials. These blended fibers have favorable properties reinforced that are required to meet the specific needs, for example desirable mechanical properties, increased hydrophilicity, increased biodegradation rate and reduced toxicity level (Park et al 2006). Recently, mixing of bioactive natural substances in the form of polymers or extracts has gained significant interest among the global research community. 1.3 BIOACTIVE NATURAL SUBSTANCES AND ITS THERAPEUTIC APPLICATIONS The term bioactive refers to a material or structure that has positive effects on the living cells in vitro and/or in vivo, due to the presence of certain bioactive substances. The major advantage of using natural bioactive substances is that they are similar to materials which are familiar to the body systems and this field of study is named as biomimetics

3 3 (Zhang et al 2007). Several natural bioactive substances of animal origin (collagen, fibrin, fibrinogen, chitin, chitosan, elastin, hyaluronic acid, chondroitin sulphate, gelatin, silk), bioceramics (bioglasses, tricalcium phosphate) and herbal extracts (Ocimum sanctum, Azardirchata indica, Curcuma longa, Syzygium aromaticum, Allium sativum, Zingiber officinalis) have been extensively studied by various researchers (Zhang et al 2007, Upadhyay & Shoeb 2012). The properties of some of the natural bioactive substances considered for the present study are briefly discussed below. Silk is a natural fibrous protein secreted by lepidoptera larvae such as silkworms and spiders. Silk fibers are composed of two main proteins: sericin and fibroin. The primary structure of fibroin protein contains highly rich glycine (Gly-Ser-Gly-Ala-Gly-Ala) n amino acid sequence which imparts tight packing of beta sheets that contribute to the rigid structure and tensile strength of silk fibroin. A combination of mechanical stability, biocompatibility and slow degradability makes fibroin potential candidate in various fields including biomedicine and textile manufacture (Altman et al 2003). Aloe vera, a medicinal plant that has been used for centuries especially for wounds, burns, insect sting and skin inflammation. Aloe vera contains bioactive phytochemicals such as acetylated mannans, polymannans, anthraquinone C-glycosides, anthrones, emodin and various lectins. The presence of saponin makes it an antibacterial agent. Acemannan and Mannose phosphate are the important components responsible for wound healing properties of Aloe vera. Emodin present in Aloe vera has anti-viral and laxative property. Further, the sterol present has anti- inflammatory activity and amino acids like phenylalanine and tryptophan reduces inflammation when administered (Vogler & Ernst 1999). The versatile uses and various

4 4 therapeutic properties of Aloe vera have attracted its application towards tissue engineering. Cissus quadrangularis is a medicinal plant used mainly for obesity, diabetes, metabolic syndrome and cardiovascular problems. It has also been used for bone fractures, weak bones (osteoporosis), scurvy, cancer, upset stomach, hemorrhoids, peptic ulcer disease, painful menstrual periods, asthma, malaria and pain. Cissus quadrangularis is also used in bodybuilding supplements as an alternative to anabolic steroids. It has antioxidant, analgesic and anti-inflammatory properties due to the presence of flavonoids especially beta-sitosterol. The stem extract contains beta carotene exhibiting strong anti-oxidant activity and the rich content of calcium, phosphorous and phytoestrogenic steroids makes it an efficient agent for bone associated disorders (Mishra et al 2010). Hydroxyapatites a bio-ceramic mineral is one of the most versatile material used clinically in a wide range of forms for applications such as bone replacement and coating material due to its similarity with natural bone mineral. It supports bone in growth and osseointegration when used in orthopaedic, dental and maxillofacial applications. It also exhibits excellent osteoconductive property with no inflammatory response and reduces healing times when used as bone substitutes (Ducheyne & Qiu 1999). In the current work Aloe vera and Cissus quadrangularis extract of plant origin, silk fibroin of animal origin and hydroxyapatite of bioceramics family are investigated for biocomposite nanofibrous scaffold fabrication for tissue engineering application.

5 5 1.4 NEED FOR BIOCOMPOSITE NANOFIBROUS SCAFFOLDS AND SCOPE OF THE PRESENT WORK In biological system, the ECM supports and maintains the cell microenvironment by providing a natural and organized web of tissuespecific nanofibers. In addition, cells in the body reside in a unique environment that is regulated by cell-cell, cell-ecm and cell soluble factors. Mimicking architecture of ECM is one of the major challenges for tissue engineering. Electrospinning can be merged with bioactive materials to generate biocomposite materials mimicking architecture of ECM and imparting bioactivity to the biologically passive synthetic polymers thereby combating the current challenges in tissue engineering (Wang et al 2013). Today electrospinning approaches to scaffold design and functionalization are beginning to expand the market for tissue engineering. Various natural polymers from animal origin were electrospun to nanofibrous scaffolds showing potential tissue engineering application. For example collagen type I with chondroitin sulphates stabilized with glutaraldehyde were fabricated by Doillon et al (2003) which showed enhanced growth, native collagen secretion and wound recovery of corneal keratocyte cells lines. Similarly in another study the 3T3 fibroblast cells grown on gelatin nanofibers showed spindle like morphology similar to normal cell morphology in a 3D extracellular matrix (Zha et al 2012). Wnek et al (2003) first published the electrospun fibrinogen and the in vitro culture with neonatal rat cardiac cells proved to be extremely bioactive with readily migration and deposition of native collagen. Park et al (2006) fabricated biomimetic nanostructured chitin/silk fibroin scaffold which showed excellent cell attachment and spreading of human epidermal keratinocytes and fibroblast. Alpha elastin and tropoelastin nanofibrous scaffold developed by Li et al (2005) supported cell attachment, migration and proliferation of

6 6 human embryonic palatal mesenchymal cells. Though enormous nanofibrous scaffolds incorporated with bioactive natural polymer from animal origin are developed only limited tries on plant extract incorporated biocomposite nanofibrous scaffolds have been investigated. Traditional medicine is in vogue and practiced for centuries in our country. However, only limited attempts have been made to combine traditional knowledge with nanotechnology. The combination of bioactive extracts from plants along with electrospun fibers has been attempted by few researchers but the evaluation of matrices with different cell lines is yet to be explored. As a preliminary effort Tecomella undulata extract incorporated nanofibrous dermal wound dressing matrix was developed by the author that gave satisfactory results, which has encouraged further approach towards tissue engineering applications of herbal extracts for the current study. In the present study electrospun nanofibrous scaffolds were developed by incorporating bioactive extracts of Aloe vera and Cissus quadrangularis along with silk fibroin and hydroxyapatite to generate a biocomposite nanofibrous scaffold of diverse origin for skin and bone tissue engineering. This dissertation is mainly focussed on analysing biological performance of the above mentioned bioactive substances with more emphasis on herbal extract incorporated matrices as scaffolds. 1.5 OBJECTIVES OF THE CURRENT RESEARCH Keeping in view of the discussions mentioned above, the objectives of the dissertation are framed 1. To fabricate and characterize Aloe vera incorporated PCL nanofibrous scaffolds for mice dermal fibroblast regeneration.

7 7 2. To fabricate and characterize Aloe vera and silk fibroin incorporated PLACL nanofibrous scaffolds for human dermal fibroblast regeneration. 3. To fabricate and characterize Cissus quadrangularis and hydroxyapatite blended PCL nanofibrous scaffolds for improved proliferation and mineralization of osteoblasts cells. 4. To develop and characterize Aloe vera, silk fibroin and hydroxyapatite incorporated PLACL biocomposite scaffolds for differentiation of mesenchymal stem cell to osteoblast lineage. 5. To develop and characterize surface precipitation of hydroxyapatite on Aloe vera and silk fibroin incorporated PCL biocomposite scaffolds for adipose derived stem cell differentiation to osteoblast lineage. 1.6 ORGANIZATION OF THE THESIS The thesis is organized into 8 chapters. The contents included in the chapters of the thesis are as follows. Chapter 1 introduces the subject in general and discusses the present status and need for the research work. It also gives the objectives of the study. Chapter 2 gives an extensive literature survey on the subject. This literature search provides a background and guide for the entire study. Chapter 3 describes the fabrication, characterization and importance of Aloe vera incorporated PCL nanofibrous scaffolds as biomimicking nanofibrous scaffold for skin tissue engineering. This chapter gives a detailed

8 8 investigation on biological responses in terms of proliferation, cell morphology and protein expression of mice dermal fibroblasts on the developed Aloe vera incorporated nanofibrous scaffolds at two different concentrations (5 and 10 wt. %) in comparison with PCL/collagen and PCL nanofibrous scaffolds. Chapter 4 deals with development and characterization of Aloe vera and silk fibroin incorporated PLACL nanofibrous scaffold for skin tissue regeneration. This chapter discusses the importance of synergistic effect of Aloe vera and silk fibroin through various biological characterizations on wound healing and tissue regeneration of human fibroblast. Chapter 5 describes the development and characterization of Cissus quadrangularis and hydroxyapatite incorporated PCL nanofibrous scaffolds for bone regeneration. The chapter also gives detail account on osteogenic property of Cissus quadrangularis along with hydroxyapatite in bringing enhanced proliferation and mineral deposition of human osteoblast cells. Chapter 6 discusses about the development and characterization of biomimetic nanofibrous scaffold incorporated with three bioactive substances namely Aloe vera, silk fibroin and hydroxyapatite. This chapter deals with detailed study on biological performance in terms of enhanced biomineralization and differentiation of human mesenchymal stem cells to osteogenic lineage. Chapter 7 deals with the development of surface precipitated hydroxyapatite on electrospun PCL nanofibrous scaffolds incorporated with Aloe vera and silk fibroin for bone tissue engineering. Cellular response of adipose derived stem cells and its differentiation to osteoblast cells were also discussed in this chapter.

9 9 Chapter 8 presents the summary of the entire study and various conclusions drawn from different studies. This chapter also contains technological relevance of the present research work and scope for conducting further research in this area.