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 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

Covalent immobilization of Aspergillus niger on pHEMA membrane: Application to continuous flow reactors

Poly(2‐hydroxyethyl methacrylate) (pHEMA) membrane was prepared via photopolymerization and activated with epichlorohydrin. The conidia of Aspergillus niger strains (wild type ‘NRRL‐3’ and genetically improved strain ‘NRRL‐3/2‐2A’) were covalently‐immobilized on the membranes. Uniform growth of A. niger cells on membrane surfaces was verified by SEM. The glucose oxidase (GOD) activity of the immobilized cells was determined in a continuous flow membrane reactor (CFMR) by assaying for hydrogen peroxide produced. The activity was also determined in the culture fluids of A. niger strains, freely grown in batch cultures. The CFMR was run with 0.1 mol dm−3 glucose with a fixed flow rate of 20 cm3 h−1 for 60 h during which a 10% loss of the original activity was detected. The loss of the activity with the freely cultivated mycelia was about 50% after 30 h. The GOD activity of the improved strain NRRL

PCL and PCL-based materials in biomedical applications

Biodegradable polymers have met with an increasing demand in medical usage over the last decades. One of such polymers is poly(epsilon-caprolactone) (PCL), which is a polyester that has been widely used in tissue engineering field for its availability, relatively inexpensive price and suitability for modification. Its chemical and biological properties, physicochemical state, degradability and mechanical strength can be adjusted, and therefore, it can be used under harsh mechanical, physical and chemical conditions without significant loss of its properties. Degradation time of PCL is quite long, thus it is used mainly in the replacement of hard tissues in the body where healing also takes an extended period of time. It is also used at load-bearing tissues of the body by enhancing its stiffness. However, due to its tailorability, use of PCL is not restricted to one type of tissue and it can be extended to engineering of soft tissues by decreasing its molecular weight and degradation time. This review outlines the basic properties of PCL, its composites, blends and copolymers. We report on various techniques for the production of different forms, and provide examples of medical applications such as tissue engineering and drug delivery systems covering the studies performed in the last decades