The use of biomaterials has become indispensable in modern medicine that includes primarily for the restoration of function as well as drug carriers. Biomaterials developed for bone, cartilage, ligament, tendon, skeletal muscle, dental, and other musculoskeletal applications almost always necessitate mechanical properties characterization to guarantee that they are robust enough for their in vivo functionality. In addition, mechanical conditioning often has a direct consequence on cellular behaviors such as differentiation, extracellular matrix production, migration, and proliferation. There is imperative necessity to get real-time data of tissue development in vivo in response to various biomechanical stimuli such as tension/compression, bending, torsion, and steady or dynamic fluid flow of construct that allows experimental protocol changes to be made early. In vitro characterization is unable to exhibit the tissue response to materials, instead being limited to the response of individual cell lines or primary cells taken from animals. Considering the wide and ever-increasing use of biomaterials in different fields of veterinary and medical sciences with its effective use in emerging fields, the characterization in respect to cellular response in the living system and its effect thereafter for leading a physiologic life, a comprehensive understanding have to be developed in totality. Further, implant safety such as avoidance of adverse tissue reaction and resistance to wear and corrosion are of high clinical significance for implants used in long-term clinical situations. The characterization along with related factors like histological, histomorphological, biochemical, radiological, scanning and transmission electron microscopic, fluorochrome labelling, biomechanical, micro-CT analysis, immunohistochemistry in orthopaedic and soft tissue surgery have been tried to elucidate with emphasis on in vivo applications of biomaterials. Amid various characterization parameters, histology is one of the most important tools to assess cellular reactions in the implant–tissue interface that can be carried out by both undecalcified and decalcified bone specimens. Histomorphometry can directly help in quantitative measurement (percentage) of newly formed bone in the implanted scaffold using semiautomatic image analysis software and also sometimes determines the host's vascularization. Histochemistry can be used to observe connective tissue ingrowth within the scaffold. The morphology and the proliferating cells can be evaluated by immunohistochemical technique. Biochemical markers like serum calcium, phosphorus, alkaline phosphatase, and osteocalcin help in evaluating the progress of healing and tartrate-resistant acid phosphatase for determining the osteoclasts activity. To understand the mechanisms of unusual bone remodelling, a number of different fluorescent stains like calcein green, tetracycline, alizarin red derivatives and xylenol orange have been developed to detect and quantify bone mineralization. Angiogenesis within the scaffold can be observed and quantified by angiography, osteomedullography, micro-CT, immunostaining with von Willebrand factor stain and intravital microscopy. Biomechanical testing is essential for quantitative assessment of implant integration and contact percentage between implant materials with the host tissue and can be performed by pull-out or push-out tests. Surface analysis and the interaction with bone tissue can be best detected by scanning electron microscopy. Non-invasive techniques include radiological, micro-CT analysis, densitometry study and ultrasound elasticity imaging (UEI). Radiological study helps to assess the union at the host bone–implant interfaces during the follow-up period and should be carried out at regular and calculated interval. Micro-CT is also a non-invasive technique and has great potential in characterization of biomaterials in regard to pore size and spatial distribution of newly formed bone together with quantitative information. Densitometric evaluation is helpful for estimating bone mineral content and density. UEI provides more information of scaffold degradation and tissue development. Finally, targeted delivery system needs quantitative measurements of biodistributable materials which can be best accomplished by computed tomography (CT), fluorescence imaging, inductively coupled atomic emission spectroscopy, inductively coupled plasma-mass spectrometry, micro-positron emission tomography, MRI imaging, and radiography. This chapter is primarily on hands-on experience in surgical manipulation of different biomaterials like hydroxyapatite, tricalcium phosphate, bioactive glass, metals, chitosan, as well as natural coralline hydroxyapatite. Different characterization techniques elaborated in this chapter can show a road map to the researchers, scientists, teachers and readers in this field of biomaterials to understand fundamental aspects of materials and related tissue response to the system in vivo. It can also provide clues for further research in the future towards this emerging field.
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