Preface This book covers most of the topics with latest information on bone in general and research on bone healing in particular. The book is divided into 27 chapters covering almost all aspect of orthopaedic research in small and lab animals including basic knowledge about bone; fracture types and fracture healing; bone grafts and bone substitute; internal fracture fixation; external fracture fixation; bone morphogenetic protein; transforming growth factors; role of mesenchymal stem cell in osteoinduction; fracture healing in critical sized bone defect and in very large bone defect, effect of herb and herbal product in fracture healing, role of different hormones, anabolic steroid, NSAID drug, bone wax, ultrasound in fracture healing; minimal invasive fracture repair; management of comminuted femoral and tibial metaphyseal fracture; endocrine role of fracture healing; evaluation of bone graft, ceramic biomaterials in fracture healing and physiotherapy of orthopedic patients. This book includes different research findings on application of herb, herbal product, bone graft, ceramic biomaterials, mesenchymal stem cells, and different osteoinducers in bone 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 orthopaedic research. The book contains more than 150 good quality photographs of internal fixation techniques, x-ray, undecalcified ground sections, scanning electron microscopy, 3-D CT scan, histopathogical stained sections used in different orthopaedic interventions.

Bone is an integral part of musculo-skeletal system of body. In the living animal it is bluish pink, whereas, fresh dead bone is yellowish-white in colour. It is hard and resistant to pressure but also have certain amount of elasticity. Its tensile strength is equal to the cast iron, though bone three times lighter and ten times flexible to cast iron. Number of bones: The number of bones of the skeleton varies with age and species. Number of bones in cattle-210; Horse-208; Dog-271; Fowl-166 and human-208 (207-212) Composition of bone: Bone contains different bone cells, organic and inorganic matrix and water. Organic matrix is about one-third and Inorganic matrix is two-third weight by volume. The organic material consists of 90% collagen [type-I (maximum), type V and type XII] and 10% non-collagenous glycoprotein and bone specific proteoglycans (osteocalcin, osteonectin, sialoprotein, bone phosphoproteins and small proteoglycans) and bone growth factors [TGF-beta family; Insulin-like growth factor-1, 2; bone morphogenetic protein (BMP); platelet derived growth factor (PDGF); Interleukin -1 (IL-1); Interleukin-6 (IL-6); colony stimulating factor]. Organic matrix of bone resembles the matrix of dense fibrous tissues such as tendons, ligament and joint capsule. It is not certain whether all of these proteins are synthesized by bone cells or whether they are synthesized by cells outside of the bone and then incorporated into the bone matrix. However, their presence within bone and their potential to affect the activity of bone cells suggests they have important role in controlling bone cell functions. Inorganic matrix of bone or mineral component of bone serves as an ion reservoir and gives bone of its stiffness and strength. It reserves 99% of body calcium, 85% body phosphorus and 40-60% body sodium and magnesium. Inorganic matrix of bone is composed of 85% calcium phosphate, 12% calcium carbonate, 3% of magnesium phosphate, calcium fluoride and magnesium carbonate. It also contains traces of chlorine, sodium, potassium. These minerals remain as hydroxyapatite crystal form within bone. However, bone mineral crystal is not pure hydroxyapatite, instead they contain both carbonate and acid phosphate groups (HPO4) and bone crystal does not contain OH groups. Bone matrix has such great durability and stability that it can remain essentially unchanged and retain much of its strength for centuries after death. Removal of organic matters renders the bone fragile, brittle and shattered, whereas, decalcification renders bone soft and pliable and resistant to fracture.

A. Definition A fracture is a break in the continuity of hard tissues like bone, cartilage, etc. B. Etiology (a) Extrinsic causes Direct violence Indirect violence

Fracture healing is a specialized type of a wound healing response in which the regeneration of bone leads to a restoration of skeletal integrity. Because healing of a fracture in a living organism is accomplished by cells, it can be modified by almost any endogenous or exogenous factors like nutrients, vitamins, hormones, growth factor etc. that has an influence on the metabolic function of bone cells. Fracture healing may be induced by malnutrition that involves certain nutrients, notably calcium (Ca), phosphorus (P) and vitamin D, but also iodine (I), copper (Cu), zinc (Zn), manganese (Mn), fluorine (F), silicon, vitamin A and perhaps vitamin C as well as different endocrines. The number of these nutrients and their interaction is so large that dietary intervention in fracture management is likely to be the most effective if directed at consumption of a good diet that is completely balanced for growth, maturity, old age or stress. Substitution of a good diet for a bad one is also likely to be more convenient and safer than most attempts at partial supplementation of a faulty diet in order to bring it into balance. Underfeeding a good diet decreases size-for-age of bones during growth and delayed fracture healing. Underfeeding may engender osteoporosis in older animals. Underfeeding of Ca, P and vitamin D, causes delayed fracture healing. Over feeding a balanced diet induces rapid growth of newly formed bone and sometimes-accelerated fracture healing but it also causes obesity in adults. Three major component of bone are osteogenic cells, organic matrix and inorganic matrix (mineral). The osteogenic cells include osteoblasts, osteocytes and osteoclasts, while the matrix consists predominantly of collagen and proteoglycans and constitutes approximately one-third of bone mass (on dry basis). The inorganic/mineral component that makes up approximately two-third of bone and it performs two essential functions : it serves as an ion reservoir and it gives bone most of its stiffness and strength. Bone is a reservoir for 99% of body calcium, 85% of body phosphorus and 40-60% of body sodium and magnesium. The main mineral composition of a long bone is: 85% calcium phosphate as hydroxyapatite, 12% calcium carbonate and 3% magnesium phosphate, calcium fluoride and magnesium carbonate and trace amount of chloride, potassium and sodium. For collagen synthesis, hydroxylase enzymes requires ferrous ion and vitamin C. Impaired collagen synthesis also occurs during deficiencies of vitamin D and E and zinc. The organic intercellular bone matrix is rich in mucoplysaccharide. Their synthesis involves enzymes that require manganese, zinc and vitamin A.

1. Bone grafts Bone grafting represents one of the earliest devised reconstructive approaches to the musculoskeletal system and remains among the most commonly used orthopedic procedures. Bone is the second most frequently transplanted tissue. The first recorded attempt to use bone graft is by the Dutch surgeon Job Van Meek’ren in 1668. Definition A graft is a living tissue that is transplanted to another site either within the same individual or into another individual for repair of a defect. An implant is a non viable material placed in a living system. Implants are composed of bone cement, metal or ceramic material. Grafts are structurally important for mechanical stability, but their use evolved to include the stimulation and enhancement of bone in response to injury. Indications Bone grafts are used, in general as framework, both to provide stability and to augment healing. It is used in the management of non union, delayed union, treatment of pseudoarthrosis, filling of osseous defects or cavities, arthrodesis, stabilization of spinal segments and addition of bone stock in total joint displacement.

Repairing fractures in small animal’s presence challenges to the veterinarians in their day to day service. These challenges include properly repairing fractures, each of which is unique, choosing the right fixation device, keeping an extensive inventory of costly instruments, and providing high standards of care for fracture repair. But fracture repair also gives veterinarians the opportunities to do something a little unusual in daily practice and to experience the immediate gratification of easing pain and promoting activity in injured animals. Successful management of fractures mainly depends upon early diagnosis, type of fracture, timing of surgery and selection of proper method of fixation. The fixation of fractures can be achieved either by external or internal fixation methods. An ideal method of fracture fixation is one that can maintain early anatomical reduction, minimal soft tissue trauma, rigid stability and early motion of bone and joint. Restoring a fractured bone’s normal shape is important for return to function because it contributes to normal alignment of adjacent joint surfaces. Whereas, rigid stability of fracture fixation eliminates fragment movement and reduces pain. Soft tissue trauma adjacent to fractured bone should be minimum, which play a vital role in early vascularisation and prevents infection. Finally, early return to function maintains muscular tone of the limb allows normal weight mobility and joint nutrition and uses normal weight bearing forces to maintain bone density. These can be achieved with use of internal fixation devices.

Fracture in animals occur frequently and is a concern of economic importance to the owner. The practice of keeping them as pets in households rather than in sheds or in confinements have increased the chance of encountering accidents like fall from coat, terrace, limbs being trapped in drains, being hit by vehicles, dog bites etc. leading to fracture. Fracture management in small animals/small ruminants can be successfully achieved owing to their light weight; they tolerate external coaptation relatively well and can ambulate in three limbs well in order to spare the injured limb. Fracture can be repaired successfully with regional anaesthetic techniques which they can tolerate very well. The constraint of cost effectiveness can be balanced by carefully selecting the appropriate technique according to the weight and limb involved. Many economical skeletal fixation technique have been evolved which can well fit into the budget of the owner. Knowledge of simple techniques can enhance healing in cases of bone loss and management of infection. Fracture site stabilization Fracture repair is usually elective, hence initial support and care of the fracture is necessary to prevent further trauma and complications like communication of fracture site to the exterior through wound or sinus and chances of infection. Initial support to splint the bone not only limits the movements but also supports the soft tissues from untoward consequences. Limb splinting reduces edema and pain, preserves neurovascular supply to the limb till the day of operation and helps to transport the animal safely. Fracture to limbs caudal to mid radius ulna/ tibia can safely be stabilized by splints or full limb casts. Wood, pliable bamboo or split poly vinyl chloride pipes positioned on the caudal and lateral aspects of the limbs and supported firmly with tapes or bandages can serve the purpose. Fracture proximal to mid radius ulna or tibia should not be stabilized using splits since the fulcrum effect created due to this can augment soft tissue injury. Careful assessment of the viability of the structures caudal to fracture site should be assessed prior to a decision regarding surgery, devitalized tissue hamper healing and is a subject to be considered for amputation.

In 1965, Urist demonstrates that the implantation of demineralized bone at extra skeletal sites induced de novo formation of cartilage and bone and proposed that the decalcified bone matrix possess high osteoinductive property and the inductor is a unique chemical that differentiates mesenchymal type cells (pericytes) into new bone. Urist and Starates (1971) reported that the inductor is a protein and termed it bone morphogenetic protein (BMP) . BMP is the bone induction principle. Sources: Bone morphogenetic protein isolate from bovine as well as human bone and designated as bBMP and hBMP respectively. The molecular cloning of the bone morphogenetic proteins and their subsequent expression in recombinant system, resulted recombinant bone morphogenetic protein designated as rhBMP. BMP has been extracted from dentin and bone matrix, as well as from bone tumours, by enzymatic and nonenzymatic methods. The sources of BMP, thus far explored are rabbit dentin, rat, rabbit and bovine bone, human bone, mouse or human osteosarcoma and human placenta. BMP circulates in the blood and it can be detected by BMP-radioimmunoassay. BMP levels higher in growing children (20-72 ng/ml) and patients with Paget’s disease (over 259 ng//ml) than in normal adults (14-18ng/ml). Crude and partially purified BMP has been extracted with the aid of collagenase, ethylene glycol, calcium chloride urea and Guanidine hydrochloride (GuHCL) solution. Wang et al (1988) purified BMP factor that induces bone formation> 300,000 fold from Guanidine chloride extracts of demineralized bone. As littlie as 50ng of this factor can induce the formation of cartilage and bone in an in vivo assay, though only 40µg could be obtained from 400kg of bovine bone powder.

Bone differs from other tissues not only in physio-chemical structure, but also in its extraordinary capacity for growth, continuous internal remodelling and regeneration throughout post fetal life. Bone formation is a complex process regulated by diet, vitamins, hormones and growth factors. Growth factors are polypeptides that increase cell replication and have important effects on differentiated cell function. Growth factors are initially considered as systemic agents, but current evidence indicates that they act primarily as local regulators of cell growth. The transforming growth factor-β family of proteins, which comprises polypeptide growth factors that have diverse effects on the growth, differentiation, and function of cells, is receiving considerable attention for potential clinical applications and is likely of physiological and orthopedically significance. Transforming growth factors It is one of the most important growth-promoting osteo-inductive substances, which have been identified at the site of fractures and other places. Transforming growth factors (TGFs) have been categorized into TGF-β (alpha) and TGF-β (beta). TGF-α has not been isolated from bone tissue and cannot be considered a local regulator of bone remodelling, although it is mitogenic for bone cells and stimulates bone resorption. TGF-α shared amino acid sequence, receptor-binding and functional activity with epidermal growth factors but had only weak transforming growth factors activity. Furthermore, TGF-β by itself is completely ineffective, but together with TGF-α, it potently induced growth of colonies of cell-line.

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, osteoconducive 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. So in this chapter, a triphasic 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 reported to evaluate the rBMSC proliferation and differentiation properties in vitro. The present chapter is therefore, included with the aim of assessing cytocompatibility of HASi and rBMSC in terms of cell attachment, cell morphology, cell proliferation and osteogenic differentiation.

The process of fracture healing is unique, in which damaged bone restores its original architecture but it does not form a poorly organized replacement matrix, otherwise known as scar tissue, rather it regenerates the original matrix and retains its mechanical properties. In order to repair bone defects, a combination of cells with osteogenic activity and appropriate scaffolding material can stimulate bone regeneration and its repair. A potential substitute for autologous transplantation should possess 3 elements: osteoprogenitor cells, osteoinductive factors, and an osteoconductive scaffold. Stem cells from adult tissues are attractive materials for cell 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. Bone-marrow-derived mesenchymal stem cells (BMSCs) have proven to be beneficial in bone regeneration. Bone marrow stem cells are pluripotent cells with the capability of differentiating osteoblasts so that they have been used to facilitate bone repair. Several studies have shown that seeding of cultured BMSCs on bioabsorbable implants can induce bone formation in vivo and lead to improved healing of critical-size bone defects. Mesenchymal stem cells (MSCs) because of their self replication and osteogenic differentiation capabilities are regarded as an excellent source of cells for bone tissue engineering. Autologous stem cells are not always preferable since the quality and quantity of such cells will be affected by metabolic diseases, old age, and osteoporosis. Allogenic BMSCs might be preferable to xenogenic, but they are always not readily available. In addition, allogenic MSCs have the potential for carrying some diseases. The use of non-autologous stem cells isolated from healthy donors offers a major advantage since these stem cells can be thoroughly tested and formulated into off-the-shelf medicine in advance. Major attractive advantage of BMSCs as a source of cell transplantation is their low immunogenecity. It is now well established that BMSCs are immune-privileged cells that do not elicit immune responses due to an absence of their immunologically relevant cell surface markers.

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. Calcium phosphate bone substitutes such as hydroxyapatite (HA) and tricalcium phosphate (TCP) are currently used for bone substitution in many different clinical applications such as repair of bone defects after trauma or tumour and bone augmentation in spinal arthrodesis. Although these bone substitutes are osteoconductive, they often lack the osteogenecity needed to support bone healing in large defects and are slowly degraded in the body. Studies have shown that coating of hydroxyapatite with a calcium silicate layer could encourage cell proliferation and osteogenic differentiation of human bone marrow-derived stromal cells. Silica-calcium phosphate composite in comparison to calcium phosphate-rich biomaterials has a faster resorption rate owing to greater dissolution of Si ions. The use of several growth factors have been studied in bone repair and these factors are known to play a role in differentiation of mesenchymal progenitor cells specific lineages like endothelial cells or osteoblast. Bone morphogenetic protein-2 (BMP-2) has been shown to accelerate bone healing in humans and animal models. BMP-2 acts by osteoinduction and is involved in the differentiation of mesenchymal progenitor cells into osteoblasts. The addition of recombinant human BMP (rhBMP-2) to a self-cross linkable cellulose hydrogels/biphasic calcium phosphate granules construct promotes bone regeneration in a critical-size segmental defect model of non-union in dogs. It is also reported that protein, insulin-like growth factor (IGF-1) stimulates direct migration of human mesenchymal progenitor cells (MPC) and contributes to the recruitment of MPC in bone formation and bone healing.

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), tri-calcium 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. Stem cells have therapeutic potential in the realm of orthopaedic surgery because of their capacity to self-renew and differentiate into various types of mature cells and tissues, including bone. Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of pre-implantation embryos and represent embryonic precursor cells that give rise to any cell type in the embryo. Specifically, it has been shown that ES cells can differentiate into osteogenic cells under selective culture conditions. These osteogenic cells are capable of in vitro producing bone nodules in three dimensional scaffolds.

Fracture healing is a specialized type of wound healing response in which the regeneration of bone leads to restoration of skeletal integrity. Though the process of fracture healing is usually considered to be biologically optimum; the healing of 5-10% is delayed or impaired. For management of these types of fractures, use of biological methods for the enhancement of fracture healing is essential. Furthermore, fracture-healing can be modified by almost any endogenous or exogenous factor that has an influence on the metabolic function of cells. These factors include transforming growth factor-beta, bone morphogenetic proteins, hormones, vitamins, prostaglandin and calcium containing feed supplement medicine. Keeping in view the above, in this chapter, efficacy of herbal product-Magacal in promoting osteogenesis during fracture healing in experimental animals is discussed. Magacal optimizes absorption, bioavailability and utilization of dietary calcium, phosphorus and magnesium. It is claimed to have advanced chelating agent to form organic complex chelate with dietary Ca, P and Mg, besides containing natural Vitamin D metabolites in highly bioavailability organic form, naturally protected and stable biotin and modern delivery system for optimum translocation of Ca, P and Mg ions to the sites of requirements. Clinically healthy, adult New Zealand White rabbits of either sex (10) are divided equally into two groups A and B each consisting of 5 animals. All animals are kept on standard diet and management throughout the experiment. In all animals, midshaft simple transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia. In test group (Group A), Magacal is given @5ml/animal/ day, orally from day 5-20 post fracture, whereas, in group B, no drug is given which serve as control. Antibiotic dressing of the skin wound is done daily for one week with povidone iodine and himax ointment. All the animals are administered streptopenicillin @ 0.5 gm/animal/day for 5 days postoperatively. Skin sutures are removed on the10th postoperative day. Clinically all the animals are observed for inflammation, pain and edema at the fracture site, surgical wound healing, appetite, general health, muscular atrophy of the limb, if any, at different intervals postoperatively.

During orthopaedic surgery, it is very difficult and sometimes impossible to stop the flow of blood from fractured bone. For this reason, there has been a longstanding interest in haemostatic agent for osseous tissue surgery. Bone –wax, oxidized cellulose, sanguistat, bone dust, marrow paste and combination using polyethylene glycols have been tried for osseous hemostasis, however, bone wax appears to be the most promising bone haemostatic agent and is widely used today. But, very littlie work has been done to define the local effect of bone wax on healing and osteogenesis and its eventual fate in animals. The objective of this study is thus, undertaken in this chapter to investigate the effects of bone wax on cortical bone healing. Clinically healthy adult New Zealand white rabbits (10) of either sex are divided equally in to two groups A and B, consisting of 5 animals each. All animals are kept in standard diet and management throughout the experiment. In all animals’ mid-shaft simple transverse fracture are created in both the ulnae under Xylazine-Ketamine anaesthesia. In animals of group A, bone wax packed in between two fracture ends, whereas, in animals of group B, fracture site kept as it is. Lateral and anterio-posterior radiographs are taken on days 0, 10, 20, 30 and 40 post-fracture. The radiographs are observed for progress of fracture healing and complication, if any at different interval. The specimens for histopathological study are collected from the healed site of test bone immediately after euthanizing the animals on day 40. Every specimen is consisted of both healing site and adjacent host bone. The bone sections are preserved in 10% formalin for 9-10 days and then are decalcified using Goodling and Stewart’s solution (15% formic acid, 5% formalin and 80% distilled water). Completely decalcified bone sections are then processed routinely for H&E staining. These sections are observed for the status of healing at the fracture site at cellular level.

Bone healing is an extraordinary process involving not only an inflammatory reaction and formation of callus but also reconstruction of the defect to regain its original biomechanical strength. Bone healing can be modified by variety of endogenous or exogenous factors that affect the metabolic function of bone cells. Prostaglandins (PG), a family of cycled derivation of unsaturated long chain fatty acids, are intimately associated with bone metabolism, and are important as locally active agents with significant effects on bone and bone cells in vitro. In some experimental studies, it is suggested that the local increase of prostaglandin in response to trauma may act to stimulate differentiation and proliferation of osteoprogenitor cells during fracture healing, whereas, specific prostaglandin antagonists, indomethacin, inhibited fracture healing. The objective of this chapter is to investigate the effects of locally (defect-site) administered prostaglandin (PGE2) on cortical bone healing in rabbit model. Clinically healthy adult New Zealand White rabbits (10) of either sex are divided equally into groups A and B, consisting of 5 animals each. They are kept on standard diet and management throughout the experiment. In all the animals, mid-shaft single transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia. In test group A, 0.1 ml (25 µg) prostaglandin (PGE2) is injected at fracture site on alternate day for 30 post operative days, whereas, group B is served as control. In both the groups lateral and antero-posterior radiographs are taken on days 0, 10, 20, 30 and 40 postoperatively to observe progress of fracture healing and complication, if any at different intervals.

The action of the gonadial hormone is not confined to the growth and development of the sexual organs alone, but the normal body growth as well as the growth of the skeleton are to a great extent influenced by these hormones. Of the female gonadial hormones, the action of estrogen and progesterone on skeletal growth has not been studied in detail. Some reports implicate estrogen deficiency in post menopausal osteoporosis and shows that estrogen replacement therapy can help to prevent that post menopausal stage of accelerated rate of bone loss. It’s reported that the progesterone treatment play a significant role in regulating cortical bone cell activity and as such, suggest that progesterone may play a part in the prevention and slowing down of cortical bone loss in females following menopause or oophorectomy. The effect of gonadotropic hormone on bone healing has not yet been studied in detail. The chapter is thus, undertaken to investigate the effects of gonadotropic hormone (Gn-rh)) on cortical bone healing in female rabbits. Clinically healthy adult female New Zealand white rabbits (10) are divided equally into groups A and B each consisting of 5 animals. All animals are kept on standard diet and management throughout the experiment. In all animals midshafts simple transverse fracture is created in both the ulnae under thiopental (2.5%) anesthesia. In test group (group A) 0.1 ml of Gn-rh analogue is injected intramuscularly at 3 days interval up to 30 days post operatively. Lateral and anterio-posterior radiographs are taken on days 0, 10, 20. 30. and 40-post fracture. These radiographs are observed for progress of fracture healing and complication, if any, at different intervals. The specimens for histopathological study are collected from the healed site of test bone immediately after euthanizing the animals on day 40. Every specimen consists of better healing site and adjacent host bone. The bone sections are preserved in 10% formalin for 9 to 10 days and then are decalcified using Goodling and Stewart’s solution (15% formic acid, 5% formalin and 80% distilled water). Completely decalcified bone sections are then processed routinely for H & E staining. These sections are observed for status of healing at fracture site.

India is bestowed with an enormous wealth of medicinal plants and has a rich heritage of treating human and animal ailments by using herbal medicines. However, there is need for a systemic, scientific approach to validate the authenticity of claims on plant-based medicaments. This approach has paid rich dividends in developing cheaper, safer, effective and eco-friendly remedies, which recently has gained worldwide attention. Cissus quadrangularis, Linn (Syn. Vitis quadrangularis, Wall; Heliotropium indicum, Linn) is a tender climber with fleshy quadrangular stems and grows in the hotter parts of Indian subcontinent, Sri Lanka and East Africa. In Hindi it is known as hardjore; in English as adamant creeper, bonesetter; in Ceylon as hiressa; in China as di xanh voung; in Burma as shazam-lese and in Bangladesh as harbhanga. Considering the advantage of herbal medicines, it is planned in this chapter to investigate the efficacy of Cissus quadrangularis, Linn, in one of the most commonly encountered problem of fractures in small animals. Dried ethanolic extract of Cissus quadrangularis (CQ) is obtained from 500g of dried finely powered stems using soxhlet apparatus. It is dissolved in a vanishing base to make a semi-solid paste for local application. For parental use, this extract is reconstituted with distilled water (100 mg/ml) and filtered. Twelve clinically healthy mongrel adult dogs aged 2-3 years and weighing 10-15 kg is randomly divided into two equal groups (A and B). In all animals operative site is prepared for aseptic surgery. Premedication is done with atropine sulphate @ 0.06 mg/ kg body weight subcutaneously and anaesthesia is achieved with a combination of Xylazine hydrochloride @ 1 mg/kg body wt. and Ketamine hydrochloride @ 10 mg/kg body wt. intravenously. Unilateral comminuted diaphyseal femoral osteotomy is created under general anaesthesia and the bone defect is immobilized with neutralization bone plating (AO/ASIF technique). All the animals are administered 5 mg/kg b wt Enrofloxacin intramuscularly for 5 d and 1 mg/ kg b wt Diclofenac sodium injection intramuscularly for 5 d postoperatively. Skin sutures are removed after 10 days. In the animals of group B, in addition to the postoperative treatment, the ethanolic extract of the herb, Cissus quadrangularis is applied on the skin twice daily along with subcutaneous injection (50 mg/ kg body wt.) on every alternate day for 20 d postoperatively.

Bone being a dynamic tissue undergoing constant biological changes, organic and mineral phases of fracture healing need an optimum general body metabolism and even a slight deviation from normal condition of the body is likely to cause considerable alterations in the healing process. Facture healing can be modified by variety of endogenous or exogenous factors that affect the metabolic function of cells. These factors include transforming growth factor beta, bone morphogenetic proteins, hormones, vitamins and prostaglandins. Anabolic steroids have the property of preventing the destruction and elimination of nitrogenous substances and minerals from the body. They help, thus, to have a positive nitrogen and mineral balance which is essential for healing and regeneration of any kind of tissue in a living organism. This chapter is undertaken to observe the effect of anabolic steroid (Durabolin) on fracture healing. This study is conducted on ten clinically healthy adult New Zealand White rabbits of either sex divided into two equal groups (X and Z) consisting five animals each. All the animals are kept in standard diet and management throughout the experiment. In all animals, midshaft segmental defect (1cm) are created in both the ulnae under thiopental (2.5%) anaesthesia. In test group (Gr X), 10mg of Nandrolone phenyl propionate (durabolin) is injected intramuscularly in 5 days interval at 7 occasions. Lateral and anterio-posterior radiographs are taken on days 0, 7, 14, 21, 28 and 35 postoperative days. The radiographs are observed for progress of fracture healing and complications, if any, at different intervals. Inflammation at the fracture site is increased upto 4th postoperative days in all the animals of both groups, and then the limb is normal in appearance by day 7. Surgical wound healing is by first intention in all the animals. Earliest return of weight bearing is seen in animals of test group followed by control. Body weight and food intake are increased two-fold in the test group of animals.

The action of the sex hormone is not confined to the growth and development of the sex organ alone, but the normal body growth as well as the growth of the skeleton are to a great extent influenced by these hormones. It is well documented that following oopherectomy, female experience a “rapid phase” of bone loss for the next few years. Of the female gonadial hormones, the action of estrogen on skeletal growth has been studies by various workers and shows that estrogen replacement therapy can help to prevent the postmenopausal stage of accelerated rate of bone loss. The effects of progesterone on bone healing or bone growth has not yet been studies in detail. The objective in this chapter is thus undertaken to investigate the effects of progesterone supplement on cortical bone healing in spayed rabbits. The study is conducted on ten clinically healthy female adult New Zealand White rabbits divided into two equal groups (A and B) consisting of five animals each. All animals are spayed and kept in standard diet and management throughout the experiment. In all animals, midshaft simple transverse fractures are created in both the ulnae under thiopental (2.5%) anaesthesia. In test group (gr A), 1mg (=1ml) of progesterone is injected subcutaneously in alternate days for upto 35 post fracture days. Lateral and anterio-posterior radiographs are taken on days 0, 7, 14, 21, 28 and 35 post-fracture. These radiographs are observed for progress of fracture healing and complications, if any, at different intervals.

Bone healing can be modified by variety of endogenous or exogenous factors that affect the metabolic function of bone cells. The Food and Drug Administration is approved the use of low-intensity ultrasound for the accelerated healing of fresh fractures in October 1994 and for the treatment of established nonunion in February 2002. The first regulatory approval is based primarily upon two rigorous, double - blind, placebo controlled clinical trials, which shows that the rate of healing of fresh fracture is accelerated by treatment with ultrasound. Therapeutic ultrasound has been able to improve functional activity, reduce post-ambulatory period, alleviate suffering and restore the animal’s health early. It has been used frequently for acceleration of healing in various musculotendinous injuries in animal. However, the role of ultrasound therapy in different dose levels in fracture healing has not been elucidated properly. In view of the above fact, this chapter is undertaken to investigate the effects of therapeutic ultrasound in two different doses, on cortical bone healing in rabbits. Twelve clinically healthy adult New Zealand white rabbits of either sex are divided equally into three groups A, B and C, and kept on standard diet and uniform management. In all animals, mid-shaft simple transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia. All the animals are administered broad-spectrum antibiotic and analgesic and surgical wounds are dressed daily with povidone iodine and antibiotic cream for 4 days postoperatively. Group A animal is received pulsed ultrasound therapy @ lW/ cm2 (1:1) whereas, group C animals is received continuous ultrasound therapy @ 3 W/cm2, starting on day 4 after the creation of fracture. Ten treatments, each consisting of 5 min duration, are given on alternate days. Group B animals are served as control. In both the test groups (A and C), the transducer head of the ultrasound machine is moved in a linear fashion along the length of the bone after applying ultrasound gel.

Standard postoperative management of animals with fractures includes the use of nonsteriodal anti-inflammatory drug (NSAID). Nonsteriodal anti-inflammatory drug inhibits inflammatory responses by blocking prostaglandin (PG) synthesis via the Cyclooxygenase pathway of arachidonic acid metabolism. However, PG is important factor in early phase of bone healing. A local increase of PG concentration is a response of bone to trauma and PG (particularly PG2) may stimulate differentiation and proliferation of osteoprogenitor cells during early bone healing. It has been reported that NSAID, such as Diclofenac acid, ibuprofen, indomethacin, aspirin, salicylic acid decreased ingrowths of bone into porous coated implants and decreased mineral content in rabbits. In view of the above fact, this chapter is undertaken to investigate the effects of one potent NSAID (Flunixin Meglumine) on fracture healing. The study is conducted in ten clinically healthy adult New Zealand White rabbits of either sex divided into two groups (A and B) consisting of five animals each. In all animals, mid-shaft simple transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia. In animals of group A, 0.25ml (12.5 mg) Flunixin Meglumine (Ilium Flunixil) is injected intramuscularly on alternate days from day 5-20 postoperatively whereas, in group B no drug is given. Lateral and anterio-posterior radiographs are taken on day 0, 10, 20, 30 and 40 post-fracture. The radiographs are observed for progress of fracture healing and complication, if any, at different interval. The specimen for histopathological study is collected from the healed site of test bones immediately after euthanizing the animals on day 40. Every specimen is consisted of both healing and adjacent host bone. The bone sections are preserved in 10% formalin for 9-10 days and then are decalcified using Goodling and Stewart’s solution (15% formic acid, 5% formalin and 80% distilled water). Completely decalcified sections are then processed routinely for H&E staining. These sections are observed for the status of healing at the fracture site, at cellular level.

Minimally invasive percutaneous osteosynthesis (MIPO), or minimally invasive fracture repair, was introduced into human orthopedic surgery during the 1990s because of several distinct advantages over traditional open reduction techniques. In MIPO, large incisions to facilitate open reduction are avoided and implants are introduced through small stab incisions in the skin. This led to advent to more biology friendly techniques. These biological techniques lay stress upon maximal preservation of blood supply around the fractured bone by minimal direct handling of the fracture environment. MIPO offer advantages which include preservation of the fracture hematoma; less surgical trauma to the surrounding soft tissues, reduced operative time therefore, decreased risk of infection and ultimately leading to indirect bone healing with abundant callus. Minimally invasive plate osteosynthesis (MIPO) is a novel technique for application of principles of biological fracture healing with a philosophy of dealing with soft tissues with utmost respect. Following advancements in implant technology and development of techniques of indirect fracture reduction this technique gained more popularity. The evolution of MIPO began with the use of bridge plating. Initially a conventional plate is applied for comminuted femoral fractures using long incisions but with preservation of the vastus lateralis muscle resulting in healing with abundant callus. Subsequently, the size of incisions got decreased, rather two small incisions are given at proximal and distal ends and plates are bridged sub muscularly over the fracture fragments. This technique causes minimal distress to soft tissues and bone, provides access to the bone through soft tissue windows, with minimal or no contact with the fracture by indirect reduction tools and leaving behind minimal foot prints.

One of the most common fractures seen in the dog is comminutuion of femoral shaft, often related to sever impact from an automobile accident or jump from a height. Comminuted fractures are characterized by one or more butterfly fragments and assorted small bone chips that are especially difficult to align accurately and stabilize completely. These complicated fractures require sophisticated repair technique that are capable to resisting compression, bending and rotational forces. Rebuilding of comminuted fractures so that all fragments are accurately aligned and gaps are eliminated is often impossible. Non-union and osteomylitis have their highest incidence in these fractures. Over the years, several techniques are tried to treat comminuted femoral fractures with different success rate and there is a lack of uniform fracture presentation. Hence, it will be difficult to compare different techniques in a particular type of fracture. The present chapter is, therefore, undertaken to evaluate radiographically and histopathologically the efficacy of three different internal fixation techniques in the management of comminuted diaphyseal femoral fracture in dogs. Clinically healthy, adult mongrel dogs (15) of either sex are randomly divided into groups A, B and C of 5 animals each. In all the animals unilateral comminuted diaphyseal femoral fractures is created under general (thiopental - 5%) anaesthesia and is immobilized with intramedullary (IM) pinning with cerclage wiring (Gr. A), neutralization bone plating (Gr. B) and neutralization bone plating with cerclage wiring (Gr. C) respectively. All the animals are administered broad spectrum antibiotic (streptopenicillin @ I g intramuscularly (dog/day) and analgesic (diclophenac sodium @ l mg/ kg b. wt. daily) and surgical wounds are dressed daily with povidone iodine and antibiotic cream for 5 days postoperatively. Lateral and oblique views of radiography are taken at the operated site just after the operation and subsequently on days 15, 30, 45 and 60 postoperatively. The radiographs are observed for status of fracture reduction and fixation, progress of healing at the fracture site and complications like fixation failure, osteomylitis etc., if any, at different intervals. Bone sections are collected from the fracture site including adjacent normal bone after sacrificing the animals on day 60. The sections are decalcified using Goodling and Stewart’s solution (80% distilled water, 15% formic acid and 5% formalin) and then processed routinely for H & E staining for histopathological studies

Among the epiphyseal and metaphyseal fractures of the different long bones, fractures of the proximal tibia are quite commonly reported and require special attention due to their proximity to the compound stifle joint. Hence, a standard fixation technique is needed to treat such fractures, which also permit early ambulation and preserve range of joint motion in addition to providing rigid fracture fixation. Different treatment modalities have been tried for fixation of metaphyseal fractures with variable results. No single method is appropriate in all circumstances and treatment indication often overlaps. Therefore, in this present chapter, an attempt has been made to evolve a standard technique for the management of such fractures. Ten clinically healthy mongrel dogs are maintained under uniform housing, feeding and management conditions, acclimatized, immunized against rabies, dewormed and randomly divided into two groups (A and B) of five animals each. In all the animals unilateral metaphyseal fracture is created in the proximal tibia under general anaesthesia with intravenous thiopental sodium (5%). In the animals of group A, the fracture is immobilized with cross intramedullary Steinmann pinning with interfragmentary wiring, whereas, single intramedullary pinning along with interfragmentary wiring is performed in the animals of group B. Antiseptic dressing of the wounds is done daily for 1 week with povidone-iodine. All the animals are administered Enrofloxacin at the dose rate of 5 mg/ kg b.wt intramuscularly daily for 5 days posoperatively. Skin sutures are removed on day 10 post-operation

Healing of fracture is a consequence of cellular behaviour in a living organism and as such can be modified by almost all endogenous or exogenous factors that affect the metabolic function of cells. These factors include transforming growth factor-β, bone morphogenetic proteins, hormones, vitamins and prostaglandins. The anabolic hormones like thyroxine and insulin accelerate bone formation in experimental animal. Hence, this chapter is proposed to evaluate the effects of thyroxine and insulin therapy in the management of tibial metaphyseal fracture in dogs. Clinically healthy, adult mongrel dogs (15) of either sex are randomly divided into three groups (A1, A2 and A3) of 5 animals each. In all the animals, unilateral metaphyseal fracture is created in the proximal tibia and then immobilized with cross intramedullary pinning with interfragmentary wiring under thiopental (5%) anaesthesia. All the animals are administered broad-spectrum antibiotic (streptopenicillin @ 1 g intramuscularly /dog/day) and analgesic (5 ml I/M daily) and surgical wounds are dressed daily with Povidone iodine and Lorexane cream for 5 days postoperatively. In group A2, thyroxine sodium tablets (0.013 mg/kg body wt, orally) and in A3, insulin injection (2 IU/kg body wt, s/c) are given on day 7 and then on every alternate day till day 45 postoperatively, at one specific time. Group A1, is remained as control, in this group no hormonal therapy is given. Craniocaudal and mediolateral radiographs of the fracture site are taken immediately after surgery and subsequent on days 15, 30, 45 and 60 postoperatively. The radiographs are observed for status of fracture reduction and fixation immediately after surgery and subsequently for the maintenance of reduction, progress of fracture healing and complication, if any, at different intervals.

Bone grafting is important in the treatment of non-unions, delayed unions, joint arthrodesis, and filling of bone cavities and the replacement of bone lost due to cyst, trauma or tumours. An ideal bone graft or substitutes should be a material that is biologically inert or bioadaptable, a source of osteogenesis, having osteoconductive property, act as a mechanical support, readily available, easily adaptable to the site in term of shape and size and replaceable by the host bone. The beneficial effect upon osteogenesis of impregnating allograft bone with autogenous red marrow or autogenous citrated plasma has been the subject of current research. It is claimed that autogenous fresh bone marrow and citrated plasma are two useful contributors for osteogenesis. Biodegradable and bioadaptable ceramics like ALCAP (Aluminum-calcium-phosphorus oxide) seem to be the most promising biomaterials for correcting bone defects, since these ceramics encourage self-replacement by endogenous bone. However, use of bone grafts and ceramics with autogenous bone marrow or citrated plasma has not been investigated so far. This chapter is undertaken to evaluate bone marrow and plasma impregnated bone allografts and ceramic implants in fracture healing in goats. Study protocol & grouping Eighteen, locally available nondescript male/female goats of 12-14 months of age are selected on the basis of their clinical status and are kept under observation for a period of one month before start of experiment. The animals are housed in their shed and are allowed to graze in the surrounding pasture. They are kept on standard feed and fodder throughout the experiment. The animals are randomly divided into six groups consisting of three animals each. Two trials of a similar experiment are performed in each goat. A total of 36 trials are conducted to evaluate different bone grafts and ceramic implants (six trials for each group with three animals each): Group A, frozen decalcified allogenic bone grafts; Group B, frozen decalcified allograft impregnated with autogenous bone marrow (composite bone graft); Group C, frozen decalcified allograft impregnated with autogenous citrated plasma (composite bone graft); Group D, ALCAP ceramics; Group E, ALCAP ceramics impregnated with autogenous bone marrow; Group F, ALCAP ceramics impregnated with autogenous citrated plasma.

Physiotherapy may be defined as the use of physical techniques like massage, exercise, heat, cold, sound waves, electric current etc for the treatment of injuries and movement dysfunction. Physiotherapy deals with the treatment of diseases by physical methods. It is used for treating injuries, strain, spasm, painful conditions, sprain of tendons, paralysis, postoperative trauma etc. It improves functioning of nerve, muscles and bones. The aim of physical therapy is the restoration of function and promotion of tissue healing by assisting normal physiological processes. The physiological response to physical therapy is its effect on the vascular supply; this in turn reproduces similar changes in deeper tissues.

References / Suggested Reading Albee F.H. (1944). Evolution of bone graft surgery. American Journal of Surgery, 63: 421-436. Aldinger G., Kusswetter G.H.W., Reis H.J. et al. (1991). Bone morphogenetic protein: a review, Internatonal Orthopaedics 15: 169-177. Albrktsson T. (1980). Repair of bone grafts. A vital microscopic and histological investigation in the rabbit. Scand J Plast Reconstr Surg, 14: 1-12. Asahina I., Sampath T.K. and Hauschka P.V. (1996). Exp Cell Res 222: 38. Bajpai P.K. (1983). Biomaterials in Reconstructive Surgery. Rubin LR (Ed), C V Mosby, St Louis, pp. 312-328. Barger Lux M.J. and Heaney R.P. (1993). Effects of calcium restriction on metabolic characteristics of premenopausal women. J Clin Endocrinology Metabolism. 76: 103-107. Bascom C.C., Sipes N.J., Coffey R.J. and Moses H.L. (1989). J. Cell Biochem 39: 25-32. Bhadane Bhagyashree K., Maiti S.K., Divya Mohan, Bag S. and Kumar N. (2018). Role of embryonic stem cell-hydroxyapatite construct with growth proteins for osteogenesis in the repair if bone defects in rabbit model. Journal of Stem Cell Research & Therapeutics, 4(4): 67-80 DOI: 10.15406/jsrt.2018.04.001117