Preface This book covers most of the topics with latest information on stem cell in general and mesenchymal stem cell (MSC) research in particular. The book is divided into 17 chapters covering almost all aspect of stem cell technology including definition of different types of stem cells, isolation of MSC from different animals, culture and characteristics of mesenchymal stem cell, osteoinduction potential of MSC, protocol for isolation of bone marrow derived MSC, fat derived MSC, embryonic stem cells (ESC), protocol for MSC passaging. This book also includes different research findings on application of bioengineered stem cell technologies in skin wound healing, burn wound healing, critical bone defect healing, large bone defect healing, corneal wound healing and in liver regeneration. The main objective of this book is to provide the latest information to meet the requirements of not only undergraduate and post graduates research scholars but also to the teachers, biologists and clinician involved in animal treatment and research. The book contains more than 150 good quality colour photographs of stem cell isolation, stem cell culture, stem cell morphology, stem cell characteristics and stem cell application in different surgical interventions.

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

References & Further Reading Agrawal S, Pittenger MF. (2005) Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 105:1815-22. Arinzeh, T. L. (2005) Mesenchymal Stem Cells for Bone Repair: Preclinical Studies and Potential Orthopedic Applications. Foot and Ankle Clinics of North America., 10: 651-665. Arinzeh, T. L., Peter, S. J., Archambault, M. P., Van Den bos, C., Gordon, S., Kraus. K., Smith, A. and Kadiyala, S. (2003) Allogeneic Mesenchymal Stem Cells Regenerate Bone in a Critical-Sized Canine Segmental Defect. J. Bone. Joint. Surg. Am., 85A: 1927. Arinzeh, T. L., Tranb, T., Mcalaryb, J. and Daculsic, G. (2005) A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation. Biomaterials, 26: 3631–3638. Atiyeh, B.S., Gunn, S.W. and Hayek, S.N. (2005) State of the art in burn treatment. World Journal of Surgery 29: 131-148. Awad HA, Butler DL, et al. (1999) Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng., 5:267-77. Badylak, S. F. (1993) Small intestinal submucosa (SIS): a biomaterial conducive to smart tissue remodeling. In: E. BELL (eds): Tissue Engineering: Current Perspectives, Birkenhäuser, Boston. pp 179-189. Badylak, S. F., Coffey, A. C., Lantz, G. C., Tacker, W. A. and Geddes L. A. (1994) Comparison of the resistance to infection of intestinal submucosa arterial autografts versus polytetrafluoroethylene arterial prostheses in a dog model. J. Vasc. Surg. 19: 465-472. Badylak, S., Liang, A., Record, R., Tullius, R. and Hodde, J. (1999) Endothelial cell adherence to small intestinal submucosa: An acellular bioscaffold. Biomaterials, 20: 2257–2263. Barrilleaux, B., Phinney, D. G., Prockop, D. J., and O’Connor, K. C. (2006) Review: Ex vivo engineering of living tissues with adult stem cells. Tissue Eng., 12: 3007–3019. Barry, F. P. and Murphy, J. M. (2004) Mesenchymal stem cells: Clinical applications and biological characterization. Int. J. Biochem Cell Biol., 36:568–584. Baur TW, Smith ST. (2002) Bioactive materials in orthopedics surgery: overview and regulatory considerations. Clin Orthop Rev., 395:11-22. Beyer, N. N. and da Silva Meirelles, L. (2006) Mesenchymal stem cells: Isolation, in vitro expansion and characterization. Hand Exp Pharmacol., pp: 249–282.