Tissue engineering of bone: the applicability of cell-based strategies

Introduction: In orthopaedic surgery the autologous bone graft is often applied. However, harvesting of this material is not without complications. Tissue engineering of bone is a promising alternative. Many authors have shown the proof of the concept and currently the first human studies are performed. However, little is actually known of the technique. Based on the available literature, we set the following study aims: 1) To investigate the role of cell viability in autologous bone grafts; 2) To investigate the role of cell viability in TE constructs; 3) To develop a labeling technique to trace cells used for TE; 4) To investigate methods to optimize bone tissue engineering; 5) To investigate the applicability of TE in clinically relevant models. Methods: All studies were performed with goat bone or bone marrow stromal cells (BMSCs). Tissue engineered constructs were made by seeding the cells on several porous ceramic scaffolds. Results Ad. 1: We investigated the role of viability in autologous bone grafts. We observed that osteogenesis was far more advanced in viable transplanted grafts, especially when implanted in the muscles (ectopically). Orthotopically, environmental bone formation probably shaded the osteogenic activity of the grafted cells. Although it is tempting to conclude that cells do survive and make bone after transplantation, this study only permits the conclusion that cell viability has an obvious effect on bone formation. This provides a rationale for cell-based bone tissue engineering. Ad. 2: The rationale for cell-based bone tissue engineering was confirmed in the goat model of ectopic bone formation in TE constructs (in the paraspinal muscles). Ad. 3: We investigated the "off the shelf" CM-Dil label and had to conclude this label was only applicable for evaluation at short implantation periods As a next step, we developed a retroviral labeling method based on the surface marker NGFR. We could proof the direct coupling between the goat BMSC's and bone formation. Ad 4: We found an advantage for constructs that were cultured for one week after seeding of the cells. When the constructs were not cultured, which makes the procedure considerably less complicated, bone formation appeared to be feasible as well. We further optimized the TE technique both with respect to the yield of bone and the ease of the procedure. By using another type of scaffold (biphasic calcium phosphate BCP), culture of the constructs appeared not to be advantageous. The method of peroperatively combining cryopreserved cells with the ceramic scaffold considerably reduced the logistics to the level of a standard elective procedure. Ad. 5: We used a bilateral iliac wing CSD model. A moderate effect of tissue engineering could be observed only at the evaluation time of 9 weeks. It is plausible that the scaffold itself was responsible for the absence of an obvious effect. Not because the scaffold is unsuitable, but on the contrary, is that potent that the majority of bone is by conduction/induction. Conclusion: Irrespective of the outcome, the studies drastically improved our knowledge on bone tissue engineering. Scientifically, the technique is intriguing and provides the opportunity to improve our knowledge on many aspects of bone formation. The answer to improvement of functionality of the tissue engineered bone may be derived from further fundamental investigations or, as often is the case in medicine, found by serendipity. The final and most important conclusion that can be drawn at the end of this thesis is: Cell-based bone tissue engineering has the potential to become an ideal autologous graft substitute

Surgical management of early-onset scoliosis: indications and currently available techniques

Early-onset scoliosis (EOS) is a heterogeneous group of spinal deformities affecting children under the age of 10 years of which the aetiology, natural history and treatment options vary considerably. In progressive EOS, treatment is based on exhausting conservative measures (casting or bracing) to halt curve progression while allowing for continuous growth of the spine and chest development. Early spinal fusion leads to loss of longitudinal spinal growth and restriction of cardiopulmonary function. In rapidly progressive curves that have failed conservative treatment a range of ‘growth-friendly’ surgical techniques have been developed to control curve deterioration. The indications and characteristics of distraction-based or compression-based methods, growth guidance and promising new techniques are discussed according to aetiology of EOS. Definitive spinal fusion remains reserved for patients ideally towards the end of their spinal growth and for short-segment treatment in congenital scoliosis

Громадська робота як чинник повсякденного життя вчителя

Treatment and reconstruction of large bone defects, delayed unions, and nonunions is challenging and has resulted in an ongoing search for novel tissue-engineered therapies. Bone morphogenetic protein-2 (BMP-2) gene therapy is a promising strategy to provide sustained production of BMP-2 locally. Alginate polymer-based nonviral gene therapy with BMP-2 plasmid DNA (pBMP-2) in constructs with multipotent mesenchymal stromal cells (MSCs) has resulted in prolonged gene expression and bone formation in vivo. To further translate this technology toward larger animal models, important issues remain to be investigated, such as the necessity of seeded cells as a target for gene therapy. For that purpose, a large animal-screening model in an orthotopic location, with fully separated chambers, was investigated. Four cylinder-shaped implants were placed in the iliac crests of ten goats. Polycaprolactone tubes around each implant allowed bone ingrowth from the underlying bone and bone marrow and ensured separation of the experimental conditions. An empty tube showed low levels of spontaneous bone ingrowth, and implantation of autologous bone indicated proper bone function with respect to remodeling and resorption. Control ceramic scaffolds were compared to scaffolds containing pBMP-2 either or not combined with seeded MSCs. Fluorochrome incorporation evaluated at 3, 6, and 9 weeks and histomorphometry at 12 weeks after implantation revealed clear differences between the groups, with pBMP-2 combined with MSCs being the most effective. The BMP-2 was demonstrated in a variety of bone-residing cells through immunohistochemistry. Further analysis indicated that multinucleated giant cells might have an important role in transgene expression. Taken together, this work introduces a large animal model for studying bone formation at multiple sites simultaneously in an orthotopic location. The model appeared robust, showed no neighboring effects, and demonstrated effectivity of combined cell and gene therapy

Схемотехническое моделирование и синтез активных СВЧ-фильтров на полевых транзисторах Шоттки

Разработаны схемы активных СВЧ-фильтров, пригодных для исполнения в виде гибридной или полупроводниковой микросхемы

The potential of spring distraction to dynamically correct complex spinal deformities in the growing child

Purpose: Current treatment of progressive early onset scoliosis involves growth-friendly instrumentation if conservative treatment fails. These implants guide growth by passive sliding or repeated lengthenings. None of these techniques provide dynamic correction after implantation. We developed the spring distraction system (SDS), by using one or multiple compressed springs positioned around a standard sliding rod, to provide active continuous distraction of the spine to stimulate growth and further correction. The purpose of this study was to determine feasibility and proof of concept of the SDS. Methods: We developed a versatile, dynamic spring distraction system for patients who would benefit from active continuous distraction. This prospective case series evaluates four patients with exceptional and progressive congenital spine deformities. Results: Four patients had a mean age of 6.8 years at surgery with a mean follow-up of 36 months (range 25–45). The mean progressive thoracic lordosis, which was the reason for initiating surgical treatment in two patients, changed from 32° lordosis preoperatively to 1° kyphosis post-operatively. During follow-up, this further improved to 32° thoracic kyphosis. In the two other patients, with cervicothorcacic scoliosis, the main coronal curve improved from 79° pre-operatively to 56° post-operatively and further improved to 42°. The mean T1-S1 spine growth during follow-up for all patients was 1.3 cm/year. There was one reoperation because of skin problems and no device-failures. Conclusion: These early results show the feasibility and the proof of concept of spring-based distraction as a dynamic growth-enhancing system with the potential of further correction of the deformity after implantation

Уровень провоспалительных цитокинов внутриматочных смывов при гиперплазиях эндометрия

Проведено вивчення змін рівня прозапальних цитокінів ІЛ -1ß, ІЛ -6 та ФНП-α в маткових змивах у жінок з різними видами гіперплазій ендометрію. Встановлено, що формування гіперплазії ендометрію супроводжується активацією прозапальних цитокінів. Найбільш виражені зміни виявлені при комплексній гіперплазії ендометрію. Запальний процес в урогенітальної системі сприяє більш вираженому зростанню рівня цитокінів в маткових змивах. Оцінка вираженості змін в рівні цитокінів маткових змивів може використовуватися в якості додаткового критерію, що характеризує гіперплазії ендометрію, для оцінки формування запальних змін в ендометрії при його гіперплазії і для оцінки прогнозу перебігу гіперплазій.Levels of proinflammatory cytokines IL-1ß, IL-6 and TNF-α in uterine lavage fluid of women with different types of endometrial hyperplasia were studied. It is established that the formation of endometrial hyperplasia is associated with activation of proinflammatory cytokines. The most intensive changes were found in complex endometrial hyperplasia. Inflammation in the urogenital system leads to more intensive increase of cytokines level in the uterine washout. Investigation of changes in cytokines levels in uterine lavage fluid can be used as an additional criterion for characteristics of endometrial hyperplasia, to assess the formation of inflammatory changes in the endometrium and for prognosis of hyperplasia

The three-dimensional coupling mechanism in scoliosis and its consequences for correction

Introduction: In idiopathic scoliosis, the anterior spinal column has rotated away from the midline and has become longer through unloading and expansion of the intervertebral discs. Theoretically, extension of the spine in the sagittal plane should provide room for this longer anterior spinal column, allowing it to swing back towards the midline in the coronal and axial plane, thus reducing both the Cobb angle and the apical vertebral rotation. Methods: In this prospective experimental study, ten patients with primary thoracic adolescent idiopathic scoliosis (AIS) underwent MRI (BoneMRI and cVISTA sequences) in supine as well as in an extended position by placing a broad bolster, supporting both hemi-thoraces, under the scoliotic apex. Differences in T4–T12 kyphosis angle, coronal Cobb angle, vertebral rotation, as well as shape of the intervertebral disc and shape and position of the nucleus pulposus, were analysed and compared between the two positions. Results: Extension reduced T4–T12 thoracic kyphosis by 10° (p < 0.001), the coronal Cobb angle decreased by 9° (p < 0.001) and vertebral rotation by 4° (p = 0.036). The coronal wedge shape of the disc significantly normalized and the wedged and lateralized nucleus pulposus partially reduced to a more symmetrical position. Conclusion: Simple extension of the scoliotic spine leads to a reduction of the deformity in the coronal and axial plane. The shape of the disc normalizes and the eccentric nucleus pulposus partially moves back to the midline

Morphologic Changes of the Intervertebral Disk During Growth

Study Design. Cross-sectional. Objective. The aim of this study was to describe morphologic changes of the annulus fibrosus (AF) and nucleus pulposus (NP) in children during growth using magnetic resonance imaging. Summary of Background Data. Little is known of intervertebral disk (IVD) maturation as opposed to degeneration, such as changes in relative AF/NP proportions and orientation during growth. Studies suggest that IVD plays a role in the etiology of pediatric spinal deformities. Therefore, understanding the morphologic development of the AF and NP during growth is key. Materials and Methods. An existing database of children aged 0 to 18 that had magnetic resonance imaging for indications unrelated to the spine were analyzed. The AF/NP were segmented semiautomatically from T1 to L5. The parameters: mean IVD height, cross-sectional area, slenderness (height/width ratio), volume (ratio), and relative position of the centroid of the NP within the IVD in three directions (x, y, z) were extracted, and compared between age, sex, and spinal level. Results. IVD height increased modestly and predominantly in the low-thoracic and lumbar spine during the first 5 to 10 years of life. Cross-sectional area and thus volume increased steadily at all levels throughout growth. IVD slenderness decreased sharply in the first years of life and remains relatively stable throughout the remainder of growth. IVDs were smaller and more slender in females, especially in the mid-thoracic spine at early adolescence. In the upper-thoracic and mid-thoracic spine the NP comprises 10% to 12% of total IVD volume during growth, this percentage increases in the low-thoracic and lumbar spine towards 20% to 25%. In the anterior-posterior direction, the position of the nucleus increasingly shifts with age, possibly in line with the developing sagittal profile of the spine. Conclusion. This study describes the development of thoracic and lumbar IVDs during growth and may be used as a reference for future studies on the role of IVD in the etiology of disk-related disorders