Andreja Vukasović

Polyester type polyHIPE scaffolds with an interconnected porous structure for cartilage regeneration

Development of artificial materials for the facilitation of cartilage regeneration remains an important challenge in orthopedic practice. Our study investigates the potential for neocartilage formation within a synthetic polyester scaffold based on the polymerization of high internal phase emulsions. The fabrication of polyHIPE polymer (PHP) was specifically tailored to produce a highly porous (85%) structure with the primary pore size in the range of 50–170 μm for cartilage tissue engineering. The resulting PHP scaffold was proven biocompatible with human articular chondrocytes and viable cells were observed within the materials as evaluated using the Live/Dead assay and histological analysis. Chondrocytes with round nuclei were organized into multicellular layers on the PHP surface and were observed to grow approximately 300 μm into the scaffold interior. The accumulation of collagen type 2 was detected using immunohistochemistry and chondrogenic specific genes were expressed with favorable collagen type 2 to 1 ratio. In addition, PHP samples are biodegradable and their baseline mechanical properties are similar to those of native cartilage, which enhance chondrocyte cell growth and proliferation.

Nanobiotechnology and bone regeneration: a mini-review

The purpose of this paper is to review current developments in bone tissue engineering, with special focus on the promising role of nanobiotechnology. This unique fusion between nanotechnology and biotechnology offers unprecedented possibilities in studying and modulating biological processes on a molecular and atomic scale. First we discuss the multiscale hierarchical structure of bone and its implication on the design of new scaffolds and delivery systems. Then we briefly present different types of nanostructured scaffolds, and finally we conclude with nanoparticle delivery systems and their potential use in promoting bone regeneration. This review is not meant to be exhaustive and comprehensive, but aims to highlight concepts and key advances in the field of nanobiotechnology and bone regeneration.

Human Mesenchymal Stem Cells Differentiation Regulated by Hydroxyapatite Content within Chitosan-Based Scaffolds under Perfusion Conditions

The extensive need for hard tissue substituent greatly motivates development of suitable allogeneic grafts for therapeutic recreation. Different calcium phosphate phases have been accepted as scaffold’s components with positive influence on osteoinduction and differentiation of human mesenchymal stem cells, in terms of their higher fraction within the graft. Nevertheless, the creation of unlimited nutrients diffusion through newly formed grafts is of great importance. The media flow accomplished by perfusion forces can provide physicochemical, and also, biomechanical stimuli for three-dimensional bone-construct growth. In the present study, the influence of a different scaffold’s composition on the human mesenchymal stem cells (hMSCs) differentiation performed in a U-CUP bioreactor under perfusion conditioning was investigated. The histological and immunohistochemical analysis of cultured bony tissues, and the evaluation of osteogenic genes’ expression indicate that the lower fraction of in situ formed hydroxyapatite in the range of 10–30% within chitosan scaffold could be preferable for bone-construct development.

Gene Therapy in Articular Cartilage Repair

The restoration of damaged articular cartilage remains one of the biggest challenges in modern clinical orthopaedics. There is no pharmacological treatment that promotes the repair of cartilage, and non-operative treatment inevitably leads to the development of premature osteoarthritis. Current treatment modalities include microfracture, transplantation of osteochondral grafts and autologous chondrocyte implantation (ACI), each having its own benefits and shortcomings. New biological approaches to cartilage repair that are based on the use of cells and molecules that promote chondrogenesis and/or inhibit cartilage breakdown offer a promising alternative to current treatment options. Chondrogenesis is a precisely orchestrated process which involves many growth factors and signaling molecules, and by modifying the local cellular environment, it is possible to enhance formation of more natural cartilage tissue within the defect. These bioactive molecules are difficult to administer effectively. For those that are proteins or RNA molecules, gene transfer has emerged as an attractive option for their sustained synthesis at the site of repair. To accomplish this task, two main strategies have been explored. The direct or in vivo approach delivers exogenous DNA directly into the joint. In this case synovial lining cells are the main site of gene transfer; depending on the vector, cells around or within the defect may also be genetically modified. During indirect or ex vivo delivery, cells are recovered, genetically manipulated outside the body, and then returned to the defect. Delivery of the genetic material to the living cell can be accomplished by use of either viral or non-viral vectors. While viral vectors are much more effective, they raise several safety concerns. Numerous preclinical animal studies have confirmed the effectiveness of these approaches in joints, and several phase I and II clinical gene therapy studies in the local treatment of arthritis provide reason for cautious optimism. This chapter will provide insight into the field of gene therapy in cartilage repair, and its potential for safe and effective clinical translation.

Normal Morphology of the Human Testis and Epididymis

The aim of this chapter is to present the histologic architecture of the human testis and epididymis to clinicians who deal with spermatozoa (micro)surgical retrieval procedures, such as testicular sperm aspiration (TESA), testicular sperm extraction (TESE), percutaneous sperm aspiration (PESA), microsurgical epididymal aspiration (MESA), and similar techniques.

“Open” Biopsy of the Testis and Testicular Sperm Extraction

Modern reproductive medicine offers new possibilities for treating infertility in men, including, among other approaches, the freezing of gametes or parts of the male or female sex gonad. In fertility practice, clinical indications for cryopreservation include storage of spermatozoa, testicular and ovarian tissues, and early embryos. Apart from freezing spermatozoa, the introduction of intracytoplasmic sperm injection into the oocyte (ICSI) in the late 1980s also created a need to freeze the testicular parenchyma. In the ICSI procedure, an oocyte is fertilized by injecting a single spermatozoon. Men with a low spermatozoa count, low spermatozoa motility, or a lack of healthy spermatozoa must provide a sample with a few viable male gametes for ICSI. Several techniques for spermatozoa retrieval from the epididymis and testis have been described and may successfully be combined with ICSI, including microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular spermatozoa aspiration (TESA), and testicular spermatozoa extraction (TESE). TESE has been used to treat cases of obstructive and nonobstructive azoospermia. Thus, frozen–thawed pieces of testicular tissue are usually used for TESE in the case of severe male infertility, including in men with azoospermia and testicular tumors. If the tumor is not too large and the surrounding testicular parenchyma is relatively preserved, one may obtain multiple testicular biopsy samples from that area and use them for TESE/ICSI.

Histologic Procedures and Testicular Biopsy Freezing

The purpose of this chapter is to describe the biopsy handling and histologic processing of testicular tissue. This chapter is intended to serve as a practical guide to performing routine procedures in the histopathologic examination and cryopreservation of the testicular biopsy specimen. Some basic staining and step-by-step laboratory procedures are described.