Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4– PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)

3D plotted PCL scaffolds for stem cell based bone tissue engineering

The ability to control the architecture and strength of a bone tissue engineering scaffold is critical to achieve a harmony between the scaffold and the host tissue. Rapid prototyping (RP) technique is applied to tissue engineering to satisfy this need and to create a scaffold directly from the scanned and digitized image of the defect site. Design and construction of complex structures with different shapes and sizes, at micro and macro scale, with fully interconnected pore structure and appropriate mechanical properties are possible by using RP techniques. In this study, RP was used for the production of poly(e-caprolactone) (PCL) scaffolds. Scaffolds with four different architectures were produced by using different configurations of the fibers (basic, basic-offset, crossed and crossed-offset) within the architecture of the scaffold. The structure of the prepared scaffolds were examined by scanning electron microscopy (SEM), porosity and its distribution were analyzed by micro-computed tomography (m-CT), stiffness and modulus values were determined by dynamic mechanical analysis (DMA). It was observed that the scaffolds had very ordered structures with mean porosities about 60%, and having storage modulus values about 1!107 Pa. These structures were then seeded with rat bone marrow origin mesenchymal stem cells (MSCs) in order to investigate the effect of scaffold structure on the cell behavior; the proliferation and differentiation of the cells on the scaffolds were studied. It was observed that cell proliferation was higher on offset scaffolds (262000 vs 235000 for basic, 287000 vs 222000 for crossed structure) and stainings for actin filaments of the cells reveal successful attachment and spreading at the surfaces of the fibers. Alkaline phosphatase (ALP) activity results were higher for the samples with lower cell proliferation, as expected. Highest MSC differentiation was observed for crossed scaffolds indicating the influence of scaffold structure on cellular activities

Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration

The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)

Biodegradable Hard Tissue Implants

Aging population and decreased physical activity due to increased life standards are two prevalent and inevitable factors that cause decrease in bone mineral mass, bone quantity, and muscle strength in the population. These consequences increase the incidence of bone fracture throughout the life of individuals. Although the bone has a great regenerative capacity compared to most other tissues or organs in the body, a proper healing of the bone requires appropriate alignment and fixation of fractured fragments throughout the process. There are different techniques and tools to provide bone substitutes with those properties. Most of the available fixation tools are made from non-eroding metals due to their inherent stiffness and toughness, the properties necessitated by the load bearing function of the skeletal system. Ideally, however, an implant should be temporary and be eliminated from the body as soon as its function is no longer necessary due to potential risks like late stage infection, bone resorption or immune reactions. For bone implants, due to the need for stabilization of fixation devices to the surrounding bone using screws or nails, removal operations may cause severe morbidity to the newly repaired fracture site. Another equally important problem with use of metal fixation devices is their superior mechanical properties that outweigh those of bone, lead the newly forming bone tissue not to be subjected to mechanical stimulation, which is a necessary requirement for bone forming machinery. Considering these problems, different biodegradable or bioerodible materials were suggested to be used in the production of temporary bone fracture fixation devices. This paper reviews the developments and trends in the field of biodegradable hard tissue implants, available materials, and their suitability to the host bone tissue.Старение населения и уменьшение физических нагрузок вследствие повышения уровня жизни являются превалирующими и неизбежными факторами, ведущими к уменьшению костной массы, минеральной составляющей костной ткани, а также снижению мышечной силы у современного человека. В результате этого увеличивается частота переломов на протяжении жизни. Хотя костная ткань обладает способностью к регенерации, сравнимой с другими тканями организма, существуют специфические особенности для восстановления ее целостности - сопоставление и фиксация отломков костей на время, необходимое для заживления. Разработаны различные технологии и средства, для придания заменителям костной ткани необходимых свойств. Большинство доступных средств фиксации изготавливают из некорродирующих металлов по причине их твердости и прочности, т.е. свойствам, обеспечивающим скелетной системе способность нести механическую нагрузку. В идеале, фиксирующий имплант должен быть временным, с возможностью удаления после восстановления нормальных функций, для предотвращения развития таких осложнений, как развитие имплантат-ассоциированных инфекций на поздних стадиях, резорбция кости или иммунные реакции. Операции по удалению фиксирующих имплантатов, вживленных в кость, нередко ведут к серьезным повреждениям новообразованной ткани костной мозоли. Другая, не менее важная проблема при фиксации металлическими средствами заключается в том, что металл обладает более высокими прочностными характеристиками по сравнению с костной тканью. Вследствие этого свойства металлических протезов механический стимул, являющийся необходимой физиологической составляющей для полноценности формирующейся кости, отсутствует. Исходя из необходимости решения данных проблем, предлагается использовать различные биодеградируемые и биоразлагаемые материалы для изготовления фиксирующих устройств при переломах кости. В статье дан обзор развития и трендов в области биодеградируемых имплантатов для твердых тканей, применяющихся материалов и их совместимости с костной тканью

A high throughput approach for analysis of cell nuclear deformability at single cell level

Various physiological and pathological processes, such as cell differentiation, migration, attachment, and metastasis are highly dependent on nuclear elasticity. Nuclear morphology directly reflects the elasticity of the nucleus. We propose that quantification of changes in nuclear morphology on surfaces with defined topography will enable us to assess nuclear elasticity and deformability. Here, we used soft lithography techniques to produce 3 dimensional (3-D) cell culture substrates decorated with micron sized pillar structures of variable aspect ratios and dimensions to induce changes in cellular and nuclear morphology. We developed a high content image analysis algorithm to quantify changes in nuclear morphology at the single-cell level in response to physical cues from the 3-D culture substrate. We present that nuclear stiffness can be used as a physical parameter to evaluate cancer cells based on their lineage and in comparison to non-cancerous cells originating from the same tissue type. This methodology can be exploited for systematic study of mechanical characteristics of large cell populations complementing conventional tools such as atomic force microscopy and nanoindentation

Biodegradable nanomats produced by electrospinning : expanding multifunctionality and potential for tissue engineering

With increasing interest in nanotechnology, development of nanofibers (n-fibers) by using the technique of electrospinning is gaining new momentum. Among important potential applications of n-fiber-based structures, scaffolds for tissue-engineering represent an advancing front. Nanoscaffolds (n-scaffolds) are closer to natural extracellular matrix (ECM) and its nanoscale fibrous structure. Although the technique of electrospinning is relatively old, various improvements have been made in the last decades to explore the spinning of submicron fibers from biodegradable polymers and to develop also multifunctional drug-releasing and bioactive scaffolds. Various factors can affect the properties of resulting nanostructures that can be classified into three main categories, namely: (1) Substrate related, (2) Apparatus related, and (3) Environment related factors. Developed n-scaffolds were tested for their cytocompatibility using different cell models and were seeded with cells for to develop tissue engineering constructs. Most importantly, studies have looked at the potential of using n-scaffolds for the development of blood vessels. There is a large area ahead for further applications and development of the field. For instance, multifunctional scaffolds that can be used as controlled delivery system do have a potential and have yet to be investigated for engineering of various tissues. So far, in vivo data on n-scaffolds are scarce, but in future reports are expected to emerge. With the convergence of the fields of nanotechnology, drug release and tissue engineering, new solutions could be found for the current limitations of tissue engineering scaffolds, which may enhance their functionality upon in vivo implantation. In this paper electrospinning process, factors affecting it, used polymers, developed n-scaffolds and their characterization are reviewed with focus on application in tissue engineering

Differentiation of BMSCs into Nerve Precursor Cells on Fiber-Foam Constructs for Peripheral Nerve Tissue Engineering

Bone marrow stem cells (BMSCs) are frequently used in nerve tissue engineering studies due to ease of their isolation and high potential for differentiation into nerve cells. A bilayer fiber-foam construct containing nanofibrous elements to house and guide BMSCs was designed as a model to study the regeneration of damaged peripheral nerve tissue and eventually serve as a nerve guide. The construct consisted of a) a macroporous bottom layer to serve as the backing and support, and for nutrient transport, and b) an electrospun, fibrous upper layer for cell attachment and guidance. Porosity and pore sizes of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) bottom layer were 85% and 5-200 μm, respectively, suitable for cell attachment and growth. Alignment of the cells is essential for cell-to-cell contact and the degree of alignment of electrospun PHBV/Collagen fibers was 11° when a frame type collector was used, while it was much higher (53°) for random fibers produced on an ordinary aluminum sheet collector. When the fibers were electrospun directly onto a PHBV foam attached on the frame type collector to create the bilayer, the degree of alignment of fibers decreased, alignment angle increased from 11° to 44°. This value did not change when the fibers were electrospun directly on the foams on the aluminum collector (53° vs 55°). A new media was designed to achieve comparable differentiation with the commercial media. It was found that the commercial Mesenchymal Stem Cell Neurogenic Differentiation Medium (PromoCell, Germany) was the better in terms of the expressions of neuronal markers nestin and β-III tubulin and the medium made in the lab with known constituents led to neuronal marker expressions very close to that with the commercial medium. Attachment and proliferation of the rBMSCs were higher on the random fiber mats, while alignment of cells was higher on the aligned fibers. In conclusion, the bilayer construct with aligned PHBV-collagen fibers on a PHBV foam was found to be more appropriate for peripheral nerve repair when used as a nerve guid

Biomaterials: from molecules to engineered tissue

Proceedings of BIOMED 2003, the 10th International Symposium on Biomedical Science and Technology, held October 10-12, 2003, in Northern Cyprus

Bone Tissue Engineering

The requirement for new bone to replace or restore the function of damaged or lost bone is a major clinical and social need. Bone tissue engineering has been considered as the alternative strategy to produce artificial bone grafts. The strategy of the method is to combine progenitor or mature cells isolated from desired cell source with biodegradable scaffolds to produce 3-D viable artificial bone in the laboratory conditions. Incorporation of growth factors that are regulators of cellular activities in vivo into the construct would protect these fragile molecules from degradation while sustaining their local concentration over a given period of time at the target site. Therefore, activities have been concentrated on the development of multi functional tissue engineering scaffolds capable of delivering the required bioactive agents to initiate and control cellular activities. This article reviews the recent developments in the production of functional artificial bone constructs via tissue engineering technique. [Archives Medical Review Journal 2010; 19(4.000): 206-219