eChapters

15.1 Introduction

Statistics is mostly used as a tool for assessing the capability of the system. Statistics refers to facts shown in numbers. Modern statistical methods and data are being found increasingly useful in research in different fields such as hydrology, biology, water quality and forestry etc. Statistics when used effectively becomes so intertwined in the whole fabric of the subjects to which it is applied. Statistics assists in planning the initial observations, organizing them and formulating hypothesis from them and in judging whether the new observations agree sufficiently well with the predictions from the hypothesis. In statistical methods, there is an operation of multiple causes and the investigator has only limited control over there. But statistical methods are found extremely useful in predicting the consequences of a situation. It requires skill for acquiring and handling data. There is a methodology to deal with the data and analysis of data is based on scientific and engineering strategies (Ramakrishnan, 1995).

In a huge rainfall data over a period of 300 years, it is not possible to observe all the items. Statistical methods are developed to estimate the error or deflection from the true or real value when we are taking inference from a sample for a hydrological data.

In the modern world where life sciences is getting more and more importance. To meet our environmental problems and energy requirements due to rapid industrialization and population growth, scop of life science has taken its new shape. Based on statistical analysis, new field like environmental sciences, hydrology, genetic engineering, biotechnology and biosociology have been evolved. Experiments about crop yields with different types of fertilizers and different types of soils or the growth of animal life and plant life under different environmental conditions and diets are very often designed and analyzed according to statistical method. Even in the field of medicine and public health, statistical methods are used for testing the efficacy of new medicine and method of treatments. As a matter of fact, for a research worker in any field, which is concern with numerical results, a study of statistical method is not only useful but also necessary. Hence it can be rightly by remarked “Science without statistics bears no fruit, statistics without science have no root”.

16.1 Conservative Technology

Recycling technology being practiced by ancient times by the people who used for municipal industrial effluent. In India the people in rural area, they collected cow dung in a pit. They applied a layer of soil on the cow dung to composting. After three to four months period, they used as manure for the agricultural fields. In a household waste, they collected as organic waste from a kitchen, store a pit allow it for decompose for conversion of compost. People who are residing in house constructed in a plot, they used to generate industrial effluent in a pit and cultivate a kitchen garden like vegetable crops and banana trees etc. in ancient times, the amount organic waste is limited and self purification is taken in small amount of organic waste therefore no organic pollution occurs in rural India.

17.1 Introduction

Bioenergy is an array of energy produced by biomass (organic matter) including liquid, solid, and gaseous fuels, electricity, heat, steam, biochemicals and materials. Biofuel is any plant derived organic matter available on a renewable basis, including dedicated energy crops and trees, agricultural food and feed crops, agricultural crop wastes and residues, wood wastes and residues, aquatic plants, animal wastes, municipal wastes, and other waste materials. An organic matter such as wood, plants, residues from agriculture or forestry, and the organic component of municipal and industrial wastes that can now be used as an energy source. Today, many bioenergy resources are replenished through the cultivation of energy crops, such as fast-growing trees and grasses, called bioenergy feedstocks.

18.1 Introduction

Recycling of industrial wastewater is not only environmentally beneficial, but it is typically more cost effective than conventional treatment and discharge. Through application of stream segregation and appropriate treatment technologies, high purity water is produced for continuous reuse where by metals are recovered and solid wastes reduced. Recycling of industrial effluent by chemical treatment includes many separation methods for getting pure waters. It is very essential to discuss about reverse osmosis, filtration, adsorption evaporation etc. This chapter includes the discussion about the methodology of chemical treatments.

18.2 Conventional Treatments

The oldest and most widely applied method of wastewater treatment involves the addition of chemicals and applications of aerators etc. to alter the make-up constituents of a waste. Although generally downsized, the process continues to play an important role in newer systems used today. Automated controls and advanced instrumentation have improved this technology in recent years. (Tomer et.al., 1997)

19.1 Introduction

The combination of two separate processes whereby water is lost on the one hand from the soil surface by evaporation and on the other hand from the tree by transpiration is referred to as evapotranspiration (ET).

19.2 Evaporation

Evaporation is the process whereby liquid water is converted into water vapour (vaporization) and removed from the evaporating surface (vapour removal). Water evaporates from a variety of surfaces, such as lakes, rivers, pavements, soils and wet vegetation. (Penman, 1963).

Energy is required to change the state of the molecules of water from liquid to vapour. Direct solar radiation and, to a lesser extent, the ambient temperature of the air provide this energy. The driving force to remove water vapour from the evaporating surface is the difference between the water vapour pressure at the evaporating surface and that of the surrounding atmosphere. As evaporation proceeds, the surrounding air becomes gradually saturated and the process will slow down and might stop if the wet air is not transferred to the atmosphere. The replacement of the saturated air with drier air depends greatly on wind speed. Hence, solar radiation, air temperature, air humidity and wind speed are climatological parameters to consider when assessing the evaporation process.

Where the evaporating surface is the soil surface, the degree of shading of the tree canopy and the amount of water available at the evaporating surface are other factors that affect the evaporation process. Frequent rains, irrigation and water transported upwards in a soil from a shallow water table wet the soil surface. Where the soil is able to supply water fast enough to satisfy the evaporation demand, the evaporation from the soil is determined only by the meteorological conditions. However, where the interval between rains and irrigation becomes large and the ability of the soil to conduct moisture to pear the surface is small, the water content in the topsoil drops and the soil surface dries out. Under these circumstances the limited availability of water exerts a controlling influence on soil evaporation. In the absence of any supply of water to the soil surface, evaporation decreases rapidly and may cease almost completely within a few days.

20.1 Introduction

High levels of energy and materials consumption in industrial countries are the driving force behind the decline in virtually all major life support systems on Planet Earth. Over the last decade an increasing number of scientists and other thoughtful people have come to conclude that modern levels of materials and energy consumption are having a destabilizing influence on the world’s ecosystems.

Energy consumption contributes directly to climate change by adding carbon-based molecules to the atmosphere in excess of naturally occurring amounts. Carbon molecules, primarily carbon dioxide from burning petroleum products, trap radiant heat and keep it from escaping from the Earth’s atmosphere. The resulting warming of the air is changing our global climate.

Materials consumption contributes indirectly to climate change because it requires energy to mine, extract, harvest, process, and transport raw materials, and more energy to manufacture, transport and, after use, dispose of products.

All the materials used in products, only 1% is used in products ‘durable’ enough to still be in use six months later, according to industrial ecologist Robert Ayres. This wasteful consumption of materials wreaks havoc on our land, water resources and their ecosystems. What’s seldom appreciated is that it also wreaks havoc on our atmosphere and contributes to climate change. Waste prevention and recycling are critical to stopping climate change.

21.1 Introduction

The industrial revolution, which began in the early 18th century in some countries of Europe, set in motion new forces in the environment and greatly changed the pattern of social life. Rapid industrialization and urbanization subsequently brought many changes in the environment and ecosystem. India also witnessed a phenomenon of industrial growth in the post independent era. Progress in the industrial development in Tamil Nadu during recent past is appreciable. New industries developed, existing industries expanded and new technologies are introduced.

In India, the past decades have witnessed population explosion and increase in per capita consumption of food and consumer goods. These have led to the proportionate increase in the domestic and industrial wastes. The waste is continuously increasing at an alarming rate. By the end of this century, India’s population is likely to increase from the present level of 700 million to about billion. Similarly the per capita consumption of food has been estimated to increase by 40% resulting in double production and disposal of industrial and domestic wastes (Manivanan, 2004).

Apart from the quantitative increase there will be also a qualitative change in the waste product, which are likely to contain greater amount of oil, toxic chemicals, detergents and dyes. A large number of these compounds are non-degradable and are cumulative, thus rendering the toxic products. Certain group of plants like algae and bacteria are selective in their functions and can executive certain toxic chemicals and heavy metals. The microorganism communities are helping in effective recycling of industrial effluents.

The government is taking serious efforts to keep the production of toxic substances under strict control at production centers. It carefully watches the efforts that are made to discharge the toxic waste from industries within the permissible limits and usages of toxic chemicals within the prescribed limits. The majority of rural population being uneducated is prone to excessive use of water which has become pollutant. The pollutant ultimately is drained into the natural aquatic environment, the rivers and the oceans. A part of these wastes are recycled and they again enter the food chain and cause detrimental effects to human health. Thus in nature, long and short term chain reactions are continuously in progress and these are required to be understood. Even when the best available yardsticks are applied to conserve the environment. It is a stupendous task to enforce these regulations.

In addition to production of more waste the coming years will see a qualitative change in the composition of waste. These wastes are likely to contain greater amount of toxic chemicals, detergents, non-degradable products based on hydrocarbons, and its derivatives. Large amount of wastes which are non degradable and cumulative in nature produce a series of new and complex problems. Therefore, alternative methods should develop for treatment and disposal of cumulative and nondegradable chemicals and pollutants.

1.1 Introduction to Numerical Method

There are three types by which problem / system can be solved and are analytical methods, graphical methods and numerical methods.

Analytical methods: This approach or methods are used to find the exact solution of simple problems / systems rather than complex systems. It provides the behavior and characteristic features of a particular system, but can only be used for systems which can be approximated through use of linear models. Another limitation is in the case of practical problems, consisting of complex and nonlinear equations / processes, as analytical methods cannot handle properly.

Graphical methods: This approach or methods are used to find the approximate solution of problems / systems through drawing of graphs for each of the equation and then check for the intersection of graphs. Intersection of graphs represents solution to system of equations, and no interaction or parallel lines of graphs represents no solution. This is not useful because of non-precise nature due to graphs drawing through hands, which also makes the process tedious. Another limitation is its viability for problems involving three or lesser dimensions.

Numerical methods: This approach or methods are used to find the approximate solution of problems / systems in a simpler way involving a large quantity of long arithmetic calculations, as compared to analytical methods. Numerical methods are the techniques by which mathematical problems are formulated to find the solution through arithmetic operations.

2.1 Introduction

Consider two linear equations in two variables i.e., x and y, such that equations are

4.1 Introduction

Numerical differentiation is the process of computing the function’s derivative values from the given set of numerical values when the actual form of the function is not known.

Similar to the numerical interpolation, a number of formulae for differentiation are derived in this chapter and are

1. Derivatives Based on Newton’s Forward Interpolation Formula: This is used to compute the derivative for some given x lying near the beginning of the data table.
2. Derivatives Based on Newton’s Backward Interpolation Formula: This is used to compute the derivative for a point near the end of the data table.
3. Derivatives Based on Stirling’s and Bessel’s Interpolation Formula: This is used to compute the derivative for some point lying in the middle of the data table.
4. Derivatives Based on Newton’s Divided Difference Formula: This is used to compute the derivative for some point when the data table is not equi- spaced.

5.1 Introduction

In applied mathematics the solution of problem generally consists of numbers which satisfy some kind of equation which are generally specified by numbers. But in practice it is found that these equations not always possible to express numerically or in exact decimal representative of the solution. Numerical methods are very important tools to provide practical methods for calculating the solutions of applied mathematics to a desired degree of accuracy. The wide use of electronic computers for solving problems in the fields of engineering, scientific, industry etc that further enhanced the scope of numerical methods.

The calculus of finite differences deals with the changes in the values of the function (dependent variable) due to changes in the independent variables. Again through this the relation of the values of the function is studied whenever the independent variable changes by finite jumps whether equal or unequal.

6.1 Introduction

Scientists and engineers have an experimental data of various variables in an experiment. To know the facts and to formulate related theories, they need exact relationship between such variables. So, they require the exact function which defines the outputs with introducing the inputs. For example,

Ohm’s law: which states that voltage of a circuit is proportional to the current flowing in it i.e.

7.1 Introduction

A Scottish mathematician David Gibb was firstly introduced a term ‘Numerical Integration’ in 1915. Like numerical differentiation, numerical integration is also a very useful tool for scientists and engineers especially when they have an experimental data for different variables without knowing any mathematical relationship between them. Beside it, there are also many other situations where it plays an important role for integrating the functions like:

8.1 Introduction

In the fields like Physics, Chemistry, Biology, Engineering and Economics; their laws and principles contains various parameters and their derivatives. The mathematical relationships of such parameters with their derivatives constitute the differential equations. In calculus, differential equations were firstly introduced by Newton and Leibnitz. Newton listed the three kinds of differential equations:

Stem cell research is one of the most important and promising areas of research in biological science in recent times. The stem cell technology has revolutionized modern biology. It is playing a major role for improving human as well as animal health by controlling number of incurable degenerative diseases and restoration of damage to organs via transplantation therapy. Stem cells are a specific group of cells that remain in an undifferentiated state and can be found in embryos, fetuses and adult individuals. Stem cells are primitive, undifferentiated cells present in almost every tissue that have the ability to differentiate into different types of tissue like bone, tendon, ligament, cartilage etc. Bone marrow stem cell is the ideal stem cell that is unspecialized and can undergo lineage specific differentiation into bone or blood cells under different signals or environment with new special function. Several verities of stem cells have been isolated and identified in vivo and in vitro. The two broad types of mammalian stem cells are embryonic stem cells that are found in blastocyst, and adult stem cells that are found in adult tissue, bone marrow.

Mesenchymal stem cells are multipotential cells capable of proliferation and differentiation into osteogenic, chondrogenic and adipogenic lineages both in vitro and in vivo. Minimal criteria for defining MSC according to International Society of Cell Therapy (ISCT) are (a) they must be plastic-adherent when maintained in a standard culture conditions, (b) MSC must express some surface antigen such as CD105, CD73, CD90, CD44 and must not express CD34, CD45, CD 14, CD19, and HLA-DR, and (c) MSC must differentiate in vitro into osteoblast, adipocytes and chondrocytes under specific differentiating condition.

Although traditionally MSC isolated from bone marrow, more recent reports have detailed the isolation of cells with MSC characteristics from a variety of tissues including umbilical cord blood, chorionic villi of the placenta, Wharton’s jelly, peripheral blood, fetal liver and lung, adipose tissue, skeletal muscle, periosteum, deciduous teeth, amniotic fluid, dental pulp, testis, synovium and the circulatory system. Bone marrow and periosteum sources are richest in young animals with their numbers diminishing, but still present in old age. It is estimated that MSCs represent approximately only 0.01 to 0.001% of the total nucleated cells within isolated bone marrow aspirate. The yield of MSCs from bone marrow varies from species to species. e.g., Canine =1 in 2.5 × 10⁴; Feline =1 in 3.8 × 10⁵; Murine =1 in 10.8 ×10³ to 1 in 3.45×10⁴ and Horses =1 in 4.2 × 10³. Despite this low number, they are remains a great interest, as they can be easily isolated from a small aspirate and culture-expanded through as many as 40 population doublings to significant numbers in about 8 to 10 weeks.

Bone marrow contains variety of tissues including hematopoietic lineage cells and mesenchymal stem cells (MSCs). The hematopoietic cells are the major source of the blood cells in the adult body which are regulated within the microenvironment of the stromal cells of the bone marrow. MSCs are multipotent cells and can be differentiate into various cell lineage depend upon their environment and culture condition in which they are kept. Rat bone marrow derived stem cells (rBM-MSCs) have long term self renewing and capabilities of pluripotency including osteoblast, adipocytes, chondrocytes, tenocytes, muscle cells and neurogenic cells, which make them ideal source of stem cells for regeneration of injured tissue.

Bone marrow derived mesenchymal stem cells have been isolated from different species. Each species is having different culturing and growing capacity. For example human bone marrow derived stem cells are easy to harvest and maintain in culture condition whereas rat MSCs it is more difficult compared to other species. Technical difficulties in isolation limited the number of animal experiment and its required animal transplantation model for pre clinical studies. Density gradient centrifugation, plastic adherence and immunomagnetic selection like various methods are available for isolation of MSCs from bone marrow. No appropriate methods are available for selection of suitable cells. Each method has their own pros and cons. Therefore, in this chapter, combined density gradient centrifugation and plastic adherence as easy and reliable method for isolation, culture and characterization of rat bone marrow derived mesenchymal stem cell is described.

Mesenchymal stem cells are multipotential cells capable of proliferation and differentiation into chondrogenic, osteogenic and adipogenic lineages. Minimal criteria for defining MSC according to International Society of Cell Therapy (ISCT) are (a) they must be plastic-adherent when maintained in standard culture conditions, (b) MSC must express some surface antigens such asCD105, CD73, CD90, CD44 and must not express CD34, CD45, CD 14, CD19, and HLA-DR, and (c) MSC must differentiate in vitro into osteoblast, adipocytes and chondrocytes under specific differentiating condition. 
Although MSC is traditionally isolated from bone marrow, more recent reports have detailed the isolation of cells with MSC characteristics from a variety of tissues including umbilical cord blood, chorionic villi of the placenta, Wharton’s jelly, peripheral blood, fetal liver and lung, adipose tissue, skeletal muscle, periosteum, deciduous teeth, amniotic fluid and synovium. Bone marrow and periosteum sources are richest in young animals with their numbers diminishing, but still present in old age. One of the attractive advantage of bone marrow derived mesenchymal stem cells (BM-MSCs) as a source of cell transplantation is their low immunogenecity. Recently, several studies have reported that BM-MSCs may be immune-privileged cells that do not elicit immune response due to an absence of their immunologically relevant cell surface markers. BM-MSCs also are known to inhibit proliferation of T lymphocytes, B lymphocytes, dendritic cells and natural killer cells.

The knowledge and principles from interconnected disciplines of bioengineering, material science and life sciences are combined in tissue engineering with the aim of developing a construct that can wholly or partly restore/maintain/augment the function of a damaged tissue or organ. Tissue engineering construct generally comprises cells on a suitable scaffold. The ultimate functionality of a tissue engineering construct is mainly dependent on the cell behavior on scaffolds. The proliferation and cell viability on scaffolds varies from one cell type to another and from one species to another. So it is important to investigate individual cell-scaffold combinations.

The similarity in structure and composition to bone mineral, osteoconductive properties, ability to integrate with the bony tissue and absence of immune response makes hydroxyapatite [Ca10 (PO4)6(OH)2] the choice of calcium phosphate biomaterial with only disadvantage of slow rate of resorption. On contrary, silica–calcium phosphate composite in comparison to calcium phosphate is rich biomaterials having a faster resorption rate. Studies have shown that coating of hydroxyapatite with a calcium silicate containing layer encourages cell proliferation and osteogenic differentiation of human bone marrow-derived stromal cells.

In this chapter a composite scaffold (calcium silicate, hydroxyapatite and tricalcium phosphate), namely HASi with elements in the following percentages: 66.36 % - calcium, 25.35 % - phosphorus and silicon - 8.29 % and porosity of 50 – 500 µm is undertaken to evaluate the rBMSC proliferation and differentiation properties in vitro with the aim of assessing cytocompatibility of HASi and rBMSC in terms of cell attachment, cell morphology, cell proliferation and osteogenic differentiation.

Fracture healing is a complex process in which damaged bone restores its original architecture through a cascade of molecular and cellular events. The process of fracture healing is unique, in that it does not form a poorly organized replacement matrix, otherwise known as scar tissue, but rather regenerates the original matrix and retains its mechanical properties. Normal fracture and bone repair follows a known sequence of events includes: haematoma formation, inflammation, angiogenesis, osteogenesis, and bone remodeling.

For the repair of bone defects, a tissue engineering approach would be to combine cells capable of osteogenic (i.e. bone-forming) activity with an appropriate scaffolding material to stimulate bone regeneration and repair. An ideal tissue-engineered bone substitute should possess 3 elements: osteoprogenitor cells, osteoinductive factors, and an osteoconductive scaffold, and is considered as a potential substitute for autologous bone transplantation. Tissue engineering approaches have proven very effective in bone regeneration recently, and the successful repair of bone defects has been demonstrated in larger animals like canine, goat and sheep.

Stem cells from adult tissues are attractive materials for cell therapy, gene therapy, and tissue engineering. These cells generally have restricted lineage potential when compared to embryonic stem cells, and this may be advantageous in certain therapeutic applications. Till and McCulloch in 1961 describes the presence of hematopoietic progenitor cells in bone marrow. After this, the concept of MSCs is brought to light by Friedenstein and co-workers who demonstrated the osteogenic potential of BM cells by heterotrophic transplantation.

Despite the benefits that minimally invasive surgery and osteosynthesis have brought to fracture management and bone healing, there are still many circumstances where bone healing remains challenging. Large bone defects are serious complications that are most commonly caused by extensive trauma, tumour, infection, or congenital musculoskeletal disorders. In nonunion cases repairing of bone defects with composite biomaterials as defect filler can promote bone regeneration. Currently, the gold standard for bone regeneration is use of autogenous bone graft. In order to avoid morbidity at the donor site or if large amount of autogenous bone is needed, bone substitution materials can be used.

Bone substitution materials can be combined with cells such as mesenchymal stem cells (MSCs) to increase bone formation. Bone-marrow derived mesenchymal stem cells (BMSCs) represent an attractive cell population for tissue regeneration. Bone marrow stem cells are pluripotent cells that have been used to facilitate bone repair because of their capability of differentiating into osteoblasts. Several studies on the regeneration of bone have shown that cultured BMSCs, seeded on different bioabsorbable implants are able to induce bone formation in vivo and lead to improved healing of critical-size defects.

Disruption of the cellular or anatomical continuity of the normal organ structure is known as wound. Healing involves migration, infiltration, proliferation and differentiation of several cell types like keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets which are culminate as an inflammatory response, new tissue formation and wound closure. Healing of the wound starts from the moment of the injury and can continue for varying periods of time depending on the extent of wound. The process of wound healing involves coagulation, inflammation, formation of granulation tissue, matrix formation, remodeling of connective tissue, collagenization and acquisition of wound strength.

Chitosan is a -1, 4-linked polymer of glucosamine (2-amino-2-deoxy—D-glucose) and lesser amounts of N-acetylglucosamine. It is a derivative of chitin (poly-Nacetylglucosamine) which is the second most abundant biopolymer after cellulose. Chitosan is first discovered in 1811 by Henri Braconnota, a French chemist and pharmacist. Later in the century, chitin is found in crustaceans (such as crabs, lobsters, shellfish and shrimp). Chitosan is a biodegradable polymer and it accelerates wound healing. It has been reported that chitosan permits regeneration of tissue elements in skin wounds and has positive application effects on wound healing.

The cornea is the anterior transparent part of the fibrous tunic of the globe which has both mechanical and optical function. It functions to support the intraocular contents and transmit and refract light. Histologically, the cornea is composed of an outer stratified squamous corneal epithelium and its basement membrane, an underlying collagenous stroma, a thin acellular membrane, and an inner monolayer of cells, the corneal endothelium. In dogs, the central cornea is thinner than the peripheral cornea, whereas regional differences in thickness may not exist in cats. The active pumping mechanism of corneal endothelium and the barrier function of anterior epithelium keep the cornea in a state of relative dehydration. Compromise of either of these mechanisms will allow corneal edema to develop, with a concomitant increase in corneal thickness. Corneal ulceration is one of the most common ophthalmic disorders in veterinary medicine. Corneal ulcers are classified based on depth and ease of healing. There are several phases during the corneal epithelial wound healing process, including a lag phase where cells alter their metabolic status; a migration phase to cover the bare surface; a proliferation phase and a differentiation phase, where cells stratify and re-establish multiple layers of distinct cells. Methods of surgical treatment for canine corneal ulcers include temporary tarsorrhaphy, third eyelid flap, conjunctival pedicle graft, tissue adhesive, corneo-scleral graft and transplantation of natural or synthetic material.

Full thickness burn wound will not heal spontaneously which need early excision and adequate coverage to prevent fluid loss and risk of infection. Major portion of the skin is lost during burn injury which is the active layer for protecting the entry of micro organism from outside environment. So far, meshed autologous skin graft is regarded as the gold standard for treating extensive full thickness burns. However, their frequent use may be hampered by several impediments such as limited availability, donor site morbidity, associated with additional scarring. Severe burn patients invariably lack additional skin donor site. Moreover, creation of donor site in burned patient is equivalent to second degree burn and which further increase the total body surface area affected (TBSA). In past decades, numerous biomaterials have been investigated for the use as a dermal skin substitute for burn patients but none give promising results. The innovative tissue engineered bioactive materials offer potential tissue regeneration and prevent the tissue deterioration.

The extracellular matrix (ECM) derived naturally from mammalian tissue have been successfully used as scaffold for tissue reconstruction and wound management in clinical applications. ECM can be derived from various tissue or organs of different species but most commonly, porcine small intestinal submucosa and urinary bladder have been successfully used as scaffold for reconstruction. ECM is obtained by process of decellularization that involves the use of various ionic and non ionic detergents, acids, enzymatic solutions and mechanical/physical methods.

Burn wounds are considered to be one of the most devastating kinds of trauma that affect the physical, physiological and psychological well being of an individual. Based on the depth of the affected area, burn wound is divided into three categories: first degree (superficial), second degree (partial) and third degree (full thickness). Extensive and deep burns result in scar formation and subsequent deformation of the affected part. Healing of burn wound involves several interrelated and overlapping stages such as inflammatory, proliferative and remodeling phases. The interaction of a variety of mediators, cells and extracellular matrix proteins are involved in this three phases of healing.

Burn wound therapy has been performed in various ways such as bioengineered skin grafts and administration of growth factors. Small intestinal sub-mucosa (SIS) is a resorbable xenogenic bioscaffold that has induced constructive remodeling in variety of animal models. It is unique from other acellular matrices as it contains few growth factors such as fibroblast growth factor (FGF) and transforming growth factor (TGF) that play a role in the regenerative process. Chitosan is currently proposed to be one of the most promising polymers in wound dressing development and it accelerates wound healing. Epidermal growth factor is known to stimulate growth of keratinocytes in vitro, thus it might be expected to promote wound healing.

In this chapter efficacy of Chitosan and porcine acellular small intestinal sub-mucosal (SIS) matrix seeded with bone marrow derived mesenchymal stem cells (BM-MSCs) and recombinant murine epidermal growth factor (m-EGF) on healing of full thickness burn wound in rat model is described.

The liver is an organ that plays a central role in maintaining metabolic homeostasis, in its functions of metabolism, synthesis and storage of nutrients. The cells in the liver are parenchyma cells and non- parenchyma cells. The former include hepatocytes (80% of the cell population) and the later includes endothelial cells, Kupffer cells, lymphocytes and stellate cells. Liver dysfunction or failures are having different etiologies. Liver cancer has the fifth highest cancer incidence in the world, and is the third highest cause of cancer related deaths, with resection of the liver remaining the only curative option.

Following liver injury, the repair process involves two distinct phases: a regenerative phase, in which injured liver cells are replaced with regenerated hepatocytes; and a phase known as fibroplasia or fibrosis, in which connective tissue replace normal parenchyma tissue. The process even though initially beneficial becomes pathogenic when it is not controlled appropriately. Extensive accumulation of extra-cellular matrix (ECM) components ultimately leads to cirrhosis and liver failure. Thus, the ideal strategy to treat liver injury is to generate new hepatocytes replacing damaged cells without causing excessive ECM deposition.

Bone is the only tissue in the body able to heal without microscopical scarring. On the other hand fracture healing is a slow process causing long immobilization periods with consequently high costs for fracture healing giving rise to delayed healing, fibrous healing or non-healing with subsequent problems for both, the patient and the orthopedic surgeon. Fracture healing is complex phenomenon which involves numerous cells, regulators of cell function and biochemical interactions in the repair process. The healing potential of bone, in a fracture model is influenced by a variety of biochemical, biomechanical, cellular, hormonal and pathological mechanisms. A continuous occurring state of bone deposition, resorption and remodeling facilitates the healing process.

Biodegradable and bioinert ceramic materials such as hydroxyapatite (HA), tri­calcium phosphate (TCP), aluminum-calcium-phosphate (ALCAP) ceramics provides scaffold to support the attachment and migration of newly formed bone cells into the osseous defect and also help in formation of a vascular network through the newly formed bone. Bioceramic also act as a carrier to deliver stem cells for osteogenesis.

Specific bone-inductive proteins/growth factors can induce bone formation and healing in vivo. These morphogens are therefore, to be an ideal alternative to autogenous bone grafts. These findings initiating intensive research into bone regeneration orchestrated by putative soluble signals and lead to the discovery and identification of an entirely novel family of protein initiators collectively called the bone morphogenic protein (BMPs), which belong to transforming growth factors-ß (TGF- ß) super family. These growth factors might possess a biological activity that is site and tissue specific in the induction of bone formation. Different growth factors have been demonstrated to possess osteoinductive and fracture healing properties.

Collect the femur and tibia and clean them very well from blood, tendons, muscle, skin and other tissues (using scalpel)

Cut off epi-and metaphysis from the long bones with electric/manual bone saw
Transfer the open bones to a falcon tubes containing cell culture medium/DPBS without FBS but containing 1% PS (Penicillin-streptomycin)
Transfer the bones to a Petri dish containing 5 ml medium without serum (FBS) and without antibiotic (40-50 mm Petri dish)
Remove the pteri dish to the laminar cabinet
Collect the bone marrow by flushing out to a 50 ml falcon tube (use syringes and needles to flush out the bone marrow; flush out the bone marrow with complete (10% FBS, 1% PS, 90% DMEM-LG) culture medium
Centrifugation: 300rpm, 8 min (in the meanwhile prepare 5 ml of Ficoll into a 15ml falcon tube)
Aspire the medium

1.    Collect fat tissues from euthanized animals into a Falcon tube containing DPBS + 1% PS ( for transferring lab to lab)
2.    Wash 3 times with DPBS + 1% PS
3.    Inside a laminar cabinet, transfer the fat to a Petridis (use an sterile spatula to collect the fat)
4.    Cut the fat into small (mm) pieces using scalpel and forceps or scissors
5.    Transfer the fat into a Falcon tube (eg into a 50 ml Falcon tube you can add upto 10 ml fat tissues)
6.    Wash the fat by adding approx 40 ml DPBS; Centrifugation: 430g, 10 min (without break)
7.    Transfer the fat into a fresh 50 ml Falcon tube (with the help of sterile spatula); Repeat steps 5 to 7 for 2-3 times

1.    Collect the bone marrow from iliac crest of rabbit, dog, goat and sheep; sternum of pigs by 16-18 G bone marrow biopsy needle under sedation/anesthesia and maintain strict asepsis protocol
2.    To aspirate bone marrow, negative pressure should create in the syringe by pulling back the plunger. Syringe should be first flushed with heparin before bone marrow collection
3.    Collect approximately 2.5-5 ml of bone marrow from each animal
4.    Mix the bone marrow sample with equal volume of low DMEM having 10% FBS and 1 ml of PS and then layer on 5 ml of histopaque/Ficol-Paque Plus in a falcon tube
5.    Centrifuge at 3000 rpm for 30 min
6.    Collect stem cells from the interface with the help of a micropipette
7.    Cells wash thrice with DPBS 
8.    Centrifuged at 2000 rpm for 5 min

1.    Remove the growth media
2.    Wash 2 times with warm PBS (w/o Ca and Mg)
3.    Add 5ml of trypsin /EDTA 0.35 % (Invitrogen), place at 370C in a CO2 incubator for 4-5 min
4.    Wait until cells are detached/lifted and make sure by examining on microscope
5.    Add equal volume (5ml-1:1) of growth medium as appropriate and mix with 5 strokes
6.    Transfer into 15 ml centrifuge tube
7.    Centrifuge at room temperature for 4 min in 980 rpm
8.    Remove supernatant complete but carefully without touching the cell aliquot
9.    Re-suspend in 10ml DPBS (with Ca and Mg)-5 strokes
10.    Immediately after mixing transfer 100µl of cell suspension into Casy vial, containing 10ml Casyton

The term ‘Horticulture’ is derived from two Latin words, Hortus meaning Garden and Cultura meaning Cultivation. In ancient days the gardens had protected enclosures with high walls or similar structures surrounding the houses. The enclosed places were used to grow fruits, vegetables, flowers and ornamental plants. Horticulture differs from agriculture and forestry in specializing the cultivation of garden crops. The garden crops include fruits, vegetables, flowers, ornamental trees, spices, plantations, medicinal and aromatic plants. Horticultural crops do not give food grains. The products of horticultural crops such as fruits and vegetables are highly perishable so that they are utilized in the fresh state. In general horticultural crops need more intensive cultivation (i.e. need more work) than the agricultural crops.

Special Features of Horticulture

As horticultural crops generally need intensive cultivation, they require a large input, capital, labour and technology per unit area of land.

Many cultural operations are specific to horticultural crops. The cultural operations such as propagation, fertilization, training, pruning, harvesting and marketing are skilled operations and these are specific to horticultural crops.

Mostly the horticultural products are utilized in the fresh state and are highly perishable.

Most of the horticultural crops are the rich sources of vitamins and minerals.

Gratification or aesthetic sense is an exclusive phenomenon for horticultural science.

Horticulture means the cultivation of garden plants. The garden plants are comprised of fruits, vegetables, ornamentals etc. The culture covers the operation from raising and planting of saplings to the ultimate use of the products and, as such, it is the most intensive of all the agricultural sciences. However, the concept of growing plants in garden or in an enclosure does not fit well in the changed perspective where large scale cultivation of fruits, vegetables and flowers extending over several square kilometers, in many a place nowadays, delimits the boundary of garden.

Importance of Horticulture

The importances of horticulture are established not only as a source of nutritious foods but also in many other ways which are narrated in the following points:

Social- Due to the long association, the fruits are seen to have a permanent seat in the social life of man. As a consequence, various fruits have acquired their place in art, music, literature, customs and habits of various nations of the world. The description of fruit as symbolic insertion in the poems and literatures, the use of whole fruit plant or part thereof in various social festive and public gathering, and conferring titles to individuals in the name of fruits or naming a region or whole district after the name of a fruit are nothing but an indication of their popularities and social position in various countries.

Fruits and vegetables play an important role in balanced diet. These provide not only energy rich food but also provide vital protective nutrients/elements and vitamins. Comparatively fruits and vegetables are the cheapest source of natural nutritive foods. Since most of Indians are vegetarians, the incorporation of horticulture produce in daily diet is essential for good health. Realizing the worth of fruits and vegetables in human health, Indian Council of Medical Research (ICMR) recommended the use of 120g fruits and 300g vegetables per capita per day. With the growing awareness and inclination towards vegetarianism worldwide the horticulture crops are gaining tremendous importance.

Functions of fruits and vegetables in human body

Fruits and vegetables provide palatability/taste.

Improves appetite and provides fiber to overcome constipation.

They neutralize the acids produced during digestion of proteins and fatty acids.

They improve the general immunity of human body against diseases, deficiencies etc.

They are the important source of vitamins and minerals for used in several bio-chemical reactions occur in body.

Classification is system of placing an individual or a member in various groups or to categories them according to particular plan or sequence which is in conformity with the nomenclature. From time-to-time for purposes of convenience plants or fruits have been classified in various ways that makes possible to refer them to a large number of kinds under a group name without enumerating or specifying individually. The basis of these groupings varies according to the features of fruits or growth of the fruit plants that have been under classification at the time.

Knowledge of classification of horticultural plants is very useful to the horticulturists because it serves: (1) to identity and name them, (2) to afford at least some idea of the closeness of their relationship i.e. line of descent to other kinds, (3) to suggest with what other kind they possibly may or may not be interbred or crossed, (4) to suggest the kinds with which they possibly may or may not be inter-grafted, and (5) often to suggest certain soil and the cultural requirements, climatic adaptations etc. However, the classification of fruit trees or fruits and vegetables on the basis of consumer’s rating also gained attention in recent years. The consumers’ rating is mainly based on the size, shape, nutritive value and marketability of them. Each of these classifications is quite different from the other classification or groupings.

Basically, the fruit trees, as suggested by many authors, have been classified on the basis of their botany comprising of taxonomic ancestry, morphologic features, physiological functions, adaptability etc. or on the basis of agricultural and horticultural requirements. But the consumers’ rating has not been properly covered in the earlier classification. Generally based on botanical relationship the plants are classified as under.

Soil- Soil is the upper most crust of earth surface which supports plant growth. It is defined as a three phase system in which plants grow. These phases are solid, liquid and gas and are essential. Solid part is frame which provides space for other two. This consists of minerals, clay minerals and organic matter. The soil is also a living system with millions of microbes that breakdown organic matter and builds it again.

Some points are following

Microbes are essential and survive only when soil is well aerated and rich in organic matter and devoid of waterlogged conditions.

Texture of soil depends on the size of solid particles and classified as gravel, coarse and fine sand, silt and clay.

Soils are classified according to relative distribution of these particles and there are 12 textural classes.

Likewise, arrangement of these particles is referred as structure, and both texture and structure lend soil physical properties like water holding capacity, aeration and bulk density.

Generally loamy soils and crumb structure are most preferred for fruit crops.

According to level of organic matter, soils are classified as mineral soil or organic soil and soil having more than 20% organic matter is organic soil like peat and muck.

Water

Water is one of the most important inputs essential for the production of crops. Plants need it continuously during their life and in huge quantities. It profoundly influences photosynthesis, respiration, absorption, translocation and utilization of mineral nutrients etc. Both its shortage and excess affects the growth and development of a plant directly and consequently its yield and quality.

Soil needs the application of water to:

Remove stress condition.

Release nutrients in the soil solution for absorption by plants.

Leach or wash out injurious salts from the soil.

Preparation of land for raising crops.

To maintain the temperature and humidity of the soil micro-climate and the activity of soil microbes at optimum level.

For the normal aeration and functioning of roots and shoots of the plants.

Excess water needs to be removed for the normal aeration and functioning of roots and shoots of the plants

Excess water creates unworkable soil condition.

Nutrients

The nutrients are chemical elements which are absorbed by the plants in more or less quantity to transform light energy into chemical energy and to keep up plant metabolism for the synthesis of organic materials. These materials constitute among other things, foods for humans and animals and a range of raw materials for various industrial uses. Feeding of plants with nutrients is termed as nutrition. Successful growth and production of the plants in general requires a proper supply of the 17 elements. These elements are regarded as essential to life in higher plants.

Weed

Weed in orchards reduce crop yields by competing for moisture, nutrients, light and space. They also harbour insect pests and diseases. When they become large they interfere with orchard operations. Some of weeds climb on the trees and shade the foliage. There are some weeds which are parasitic partially or completely on the host tree e.g. Striga and loranthes on mango.

Mulching

It means covering of soil surface by some organic or inorganic materials for conserving the soil moisture besides covering on the soil moisture several other advantage of mulching. The materials which are used in mulching called mulch.

Nursery

Nursery is a place where seedling, saplings or any other planting materials are raised, propagated, multiplied and sold out for planting. The prerequisites of a successful and remunerative fruit production are the availability of true-to-type, healthy and good quality planting materials. Setting up of a fruit nursery is a long term venture and requires careful planning and expertise, because mistakes committed initially cannot be rectified easily and may adversely affect the return from the investment. Thus, one should pay due attention on every aspects when nursery is to be established.

Propagation

Plant propagation means multiplication of plant by sexual or asexual method. The success in plant propagation depends on a thorough knowledge of the growth of plants, choice of suitable method and technical skill. The propagation of plants may be done either by the gametes or from the somatic cell, tissues or organs. In other words, propagation may be done by sexual methods or by asexual methods. The details of the methods are presented below.

Orchard

Establishment of orchard is a long term investment. It is require a proper planning, any mistake during the selection of site, planting distance, choice of crop/variety, quality of nursery stock etc. reflect greatly on the orchard performance and efficiency.

Planting System

Main objective of layout is maximum number of plant accommodated in per unit area without affecting the efficiency of production. If more than one fruit is included in the orchard plan, each kind should be grouped into individual block. Similarly fruit ripened in particular season should be grouped to facilitate convenience in watch and ward, harvest and post harvest handling. Some kind of fruit trees need pollinators/pollinisers for improving the fruit set.

Horticultural plants are grown for their produce like fruit, vegetable, flower, medicinal component, spices etc. Therefore, these plants should be managed in such a way that human desires for the purpose of growing them are fully satisfied in terms of quality and quantity of produce. This demands direct manipulation of plant growth itself or plant environment through various inputs. In manipulation of plant development, training and pruning are important for which our knowledge about plant development and its phenology has to be complete. These practices are important in fruit crops.

Bearing

Bearing is connotation of flowering and fruiting. When a fruit tree flowers and fruits, it is said in bearing condition. Different fruits have different bearing habits, inflorescences, types of flower, mode and medium of pollination, pollen viability and all these account a lot for the bearing of fruit plants.

Bearing habit

If one observes a mango tree, he/she may find it does produce the fruits terminally on a shoot and it citrus he/she finds the fruits always produced laterally in the leaf axils. The manner or patter in which a plant produces its fruit is known as bearing habit or fruiting habit. Bearing habits are specific to the kind of plant. In determining the bearing habit of any plant three points are to be considered.

The quantitative increase in plant body such as increase in the length of stem and root, the number of leaves etc., is referred to as plant growth whereas, the qualitative changes such as germination of seed, formation of leaves, flowers and fruits, falling of leaves and fruits is referred as development. Utilization of these substances for proper development of the plant is regulated by certain chemical messengers called plant growth substances or plant growth regulators, which in minute amounts increase or decrease or modifies the physiological process in plants. Plant growth substances are the chemical other than nutrient which required in very small amount but without which plant can not complete their physiology.

The two sets of internal factors, viz., nutrition and hormone control the growth and development of the plant. The raw material required for growth is supplied by nutritional factors which include the minerals, organic substances the protein, carbohydrates, etc.

Phytohormones: These are the hormones produced by plants which in low concentrations regulate plant physiological process. These usually move within the plants from a site of production to a site of action.

Plant growth regulators: These are organic compounds other than nutrients, which in small amounts promote, inhibit or otherwise modify any physiological process in plant.

It may be defined as any organic compounds which are active at low concentrations (1-10 ml) in promoting, inhibiting or modifying growth and development in plants. The naturally occurring (endogenous) growth substances are commonly known as plant hormones, while the synthetic ones are called growth regulators.

Plant hormones: It is an organic compound synthesized in one part of plant and translocated to other parts, wherein very low concentration causes a physiological response.

The plant hormones are identified as promoters (auxins, gibberellin, and cytokinins), inhibitors (abscisic acid and ethylene) and other hypothetical growth substances (Florigen, death hormone, etc.).

The current status of advances in fruit breeding is through the development of different scientific approaches augmented by ever-increasing knowledge and application of genetics, statistics and biotechnology. These innovative approaches very specific for a species in most of the cases, are the major tools of the fruit breeders to tackle the different objectives of fruit improvement programme at present Fruit improvement is a very slow process due to the long generation cycle varying from species to species, thereby reducing the chances of exploiting genetic recombination. Besides this factor, most of the fruit species are either highly heterozygous or polyploidy in nature. Due to this reason the asexual propagation of fruit plants is a conventional means while wide segregation occurs upon sexual reproduction. Such expectation is not unlikely because the types selected as commercial varieties are definitely to be vigorous ones, and a positive correlation exists between vigour and heterozygosity.

This discussion on breeding of fruit plants involves different aspect of the species concerned is so essential as well as fundamental that discussion of them must procede any consideration of breeding methods themselves. The plant species are grouped into two-self-pollinated and cross-pollinated. These two groups have emerged on the basis of pollination mechanism which plays significant role in the method of breeding application. To emphasize the difference in the reproductive systems of these two groups one should know the details about floral biology, mechanism of pollination control, reproduction by asexual and sexual means and self and cross-pollinated plants.

Physiological disorders are abnormalities in plants which are associated to non-pathogenic factors. A plant or its parts may show unusual growth, function or deformity. Such abnormalities are widely referred to as disorders. These may be incited by nutrients deficiency or excess, hormonal imbalance, abnormal growing conditions etc. Some commonly observed disorders have been dealt in here suitably.