eChapters

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Cassava (Syn: Tapioca) Manihot esculenta (Crantz) (2n = 2x = 36) (Hindi : Mravuli) is perennial shrub cultivated in tropics for its starchy tuberous roots. It is one of important tuber crops cultivated in tropics because of its high carbohydrate yield per unit land and labour and its adaptation to poor soil and water stress and its tolerance to dominant pest and diseases. Cassava contains cyanide, the toxic principle which limits food and feed value. In cassava the glycoside linamarin and lotaustralin are converted to hydrocyanic acid, a potent toxin, when come in contact with linamerase, an enzyme that is released from rupture of cells. Tuber peel contain significantly higher amount of glycosides than the pulp. It can be removed by cooking in water for about 5 minutes.

Sweet potato Ipomea batatas(L). Lam. is an asexually propagated vegetable. It is grown in most of the tropical and subtropical region of the earth. In USA production extended to temperate regions. Vines as well as roots are eaten or fed to live stock. It is the source of industrial starch and potential source of the alcohol. Among the major tuber crops cultivated in India sweet potato rank third after potato and cassava in area and production.

Origin: Available evidence suggests South Mexico through Central America and Northern South America as the probable center of origin. It is perennial but handled as annual. Its hardiness and drought tolerance are well recognized.

Family: Convolvulaceae, Chromosome number: 2n=90

It is the only known natural hexaploid morning glory. Most of the wild species are diploid (2n=30). Hybridization is restricted with in species because of its hexaploidy nature. Extensive variability with in species is observed. Tuber is the fleshy subterranean stem or shoot but in sweet potato tuber is a root, and hence it cannot be a tuber (common error). It is a perennial, and the storage root is capable of continued enlargement and does not mature in the sense of reaching some final size or stage of development. From the stand point of function, the root system consists of absorbing roots and fleshy storage roots.

Turnip is the root vegetable cultivated in cooler season or temperate regions of the globe. Brassica rapa L 2n = 2x = 20 is formerly known as B. campastris subsp rapifera. Swede (B. napus var. napobrassica) is directly related to turnip.
Origin: It is originated in cooler part of Europe, presumably from biennial oilseed forms. It was known to Greeks and Romans at beginning of Christian era. It was introduced to Britain from France by Romans and into North America by the early European settlers in the 17th century.

Beta vulgaris 2n = 2x=18 is an important vegetable crop in Eastern and Central Europe and it belongs to family Chenopodiaceae. It is of lesser importance in Western Europe and USA where it is known as garden beet. The swollen roots are consumed as vegetable and salad. The other types which are non-horticultural forms are known as sugar beet, mangold and fodder beet.
Origin: Leaf beet is the earliest domesticated form. It is believed to be originated from Beta maritima known as sea beet which is indigenous to Southern Europe. 
Botany: Biennial producing enlarged hypocotyl (roots) and rossette of leaves in first year and flowers and seeds in second year.

It is a popular root crop having wide range of adaptability, grown in Japan, Koria, China, India and other European countries.

Origin and Distribution: Radish was an important food in Egypt during 2000 BC.  Fossils study reveals that, it was a food as early as 2700 BC. It spread to China during 500 BC and to Japan during AD 700. Variability of crop decreased from Europe to China and from there to Japan. Recent study indicates that East of Mediterranean, most probably China was the centre of origin of the crop. This means Europe and Asia are the probable places of origin.

Carrot (Daucus carota L.) is a cool season root vegetable and temperature is the important factor affecting root shape and colour, besides soil type and moisture content of soil. It provides an excellent source of vitamin A and fiber in the diet. The genus Daucus, which include carrot, has many wild forms that grow mostly in the Mediterranean region and South West Asia. Representatives are found in Africa, Australia, & North America. For Daucus carrot L. it is generally agreed that Afghanistan is the primary center of genetic diversity and therefore the primary source for dissemination. Asia Minor, Afghanistan, North West India, Iran and Turkey are the centers of origin of carrot. Banga (1957& 1963) has provided evidence that the purple (anthocyanin) carrot together with a yellow variant spread from Afghanistan to Mediterranean area as early as 10th and 11th century. It is known in Western Europe in 14th and 15th century, China in 14th century and Japan in 17th century. The European carrot (Carotene carrot) derived from the Asiatic (anthocyonin containing) form of carrot (tropical carrot).  Afghanistan is the centre of diversity of the purple coloured carrot (anthocyanin). Integration of Asiatic and European carrots has given the present day forms. The yellow coloured (carotene rich) forms were also thought as mutants of anthocyanin carrots. The yellow mutants were traced in Asia Minor in 10 and 11th centuries, Spain in 12th century, North Western Europe in 14th century and England by 15th century.  The crop was introduced to China during 14th century and to Japan during 17th century. D. carota and D. sativus are only cultivated species of the genera with Chromosome No. 2n = 18. All forms have same chromosome number. 

Amaranthus is one of the main genera of large and taxonomically diverse group of leafy vegetables. Amaranthus is the cheapest dark green leafy vegetable because of low production costs and high yield. It is often described as poor man’s vegetable.

Origin and Distribution: The main vegetable type Amaranthus tricolor L. originated in South or South East Asia, particularly in India. Budhist monks and Muslim invaders took the crop to neighboring countries. Another vegetable type A. dubius shows diversity in Central America, Indonesia, India and in Africa. A lividus is a popular leafy vegetable in Southern and Central Europe. Amaranthus included 50 to 60 species.

Cauliflower follows cabbage in importance with regard to area and production in the world. However in India Cauliflower is more widely grown than cabbage. This crop grows at a latitude 11°N to 60°N with average temperature ranging from 5- 8°C to 25-28°C. It can stand to the temperature as low as -10°C and as high as 40°C for a few days. Italy and India are the major countries growing CF on large scale. Important cauliflower growing states in India are UP, Karnataka, West Bengal, Punjab and Bihar. It is commonly grown in Northern Himalayas and Niligiri hills in south.

Origin and Distribution: Cauliflower is thought to have been domesticated in the Mediterranean region since, the greater range of variability in the wild types of B.oleracea is found there (Nieuwhof, 1969). Wild Cole warts are still found in wild state in sea cost of England and Denmark and in the North West France and various localities from Greece to Great Britain. The Cole crops, including cauliflower and cabbage have descended from a common Kale like ancestor, the wild cabbage (B.olearcea var. sylvestris L.) which is still found in western and southern Europe and North Africa (i.e, sea coasts of England and Denmark and in North West France and various localities from Greece to Great Britain). Cyprus and area around Mediterranean courts are considered to be the primary centers of origin for Cauliflower. It is also reported that cauliflower is originated from a related species. B.cretica. As per Bosewell (1949), it originated in the islands of Cyprus from where it moved to other areas like Syria, Turkey, Egypt, Italy, Spain and North Western Europe. Cornish types originated in England fallowed by temperate types originated in Germany and Netherlands in 18th century. The present Indian tropical cauliflowers developed as a result of intercrossing between European and Cornish type. In India tropical types with resistance to high temp and high rainfall conditions have been developed. Tropical types are grown in Indian plains from May to September, followed by temperate types known as snowball cauliflower.