SEMS

Poor fixation of bone replacement implants, e.g. the artificial hip, in implantation sites with inferior bone quality and quantity may be overcome by the use of implants coated with a cultured living bone equivalent. In this study, we tested, respectively, amorphous carbonated apatite (CA)-and crystalline octacalcium phosphate (OCP)-coated discs for their use in bone tissue engineering. Subcultured rat bone marrow cells were seeded on the substrates and after 7 days of culture, the implants were subcutaneously implanted in nude mice for 4 weeks. After 7 days of culture, the cells had formed a continuous multi-layer that covered the entire surface of the substrates. The amount of cells was visually higher on the crystalline OCP-coated discs compared to the amorphous CA-coated discs. Furthermore, the amorphous CA-coated discs exhibited a visually higher amount of mineralized extracellular matrix compared to the crystalline OCP-coated discs. After 4 weeks of implantation, clear de novo bone formation was observed on all discs with cultured cells. The newly formed bone on the crystalline OCP-coated discs was more organized and revealed a significantly higher volume compared to the amorphous CA-coated discs. The percentage of bone contact with the discs was also significantly higher on the OCP-coated discs. Overall, the results suggest that a crystalline OCP coating is more suitable for bone tissue engineering than an amorphous CA coating. r

The preparation, characterization, and in vitro bone marrow cell culturing on porous PEOT/PBT copolymer scaffolds are described. These scaffolds are meant for use in bone tissue engineering. Previous research has shown that PEOT/PBT copolymers showed in vivo degradation, calcification, and bone bonding. Despite this, several of these copolymers do not support bone marrow cell growth in vitro. Surface modification, such as gas-plasma treatment, is needed to improve the in vitro cell attachment. Porous structures were prepared using a freeze-drying and a saltleaching technique, the latter one resulting in highly porous interconnected structures of large pore size. Gas-plasma treatment with CO 2 generated a surface throughout the en-tire structure that enabled bone marrow cells to attach. The amount of DNA was determined as a measure for the amount of cells present on the scaffolds. No significant effect of pore size on the amount of DNA present was seen for scaffolds with pore sizes between 250-1000 m. Light microscopy data showed cells in the center of the scaffolds, more cells were observed in the scaffolds of 425-500 m and 500-710 m pore size compared to the ones with 250-425 m and 710-1000 m pores.

Successful bone-tissue engineering (TE) has been reported for various strategies to combine cells with a porous scaffold. In particular, the period after seeding until implantation of the constructs may vary between hours and several weeks. Differences between these strategies can be reduced to (a) the presence of extracellular matrix, (b) the differentiation status of the cells, and (c) the presence of residual potentially immunogenic serum proteins. These parameters are investigated in two types of calcium phosphate scaffolds in a goat model of ectopic bone formation. Culture-expanded bone-marrow stromal cells from eight goats were seeded onto two types of hydroxyapatite granules: HA60/400 (60% porosity, 400-m average pore size) and HA70/800. Scaffolds seeded with cells and control scaffolds were cultured for 6 days in medium containing autologous or semisynthetic serum, in the presence or absence of dexamethasone. Other scaffolds were seeded with cells just before implantation in medium with or without serum. All conditions were implanted autologously in the paraspinal muscles. After 12 weeks, bone had formed in 87% of all TE constructs, as demonstrated by histology. Histomorphometry indicated significantly more bone in the HA70/800 scaffolds. Furthermore, a significant advantage in bone formation was found when the constructs had been cultured for 6 days. In conclusion, both scaffold characteristics (porosity) and TE strategy (culturing of the constructs) were demonstrated to be important for bone TE.

One of the key issues in successful application of biomaterials for tissue engineering and drug delivery is a well-characterized in vivo biodegradation behavior. Poly(lactic-co-glycolic acid) (PLGA) has been used for a wide variety of medical applications, from resorbable sutures to bone screws and microspheres for drug delivery. Degradation of PLGA in vitro as well as in vivo mainly takes place through either hydrolysis of the ester linkages and/or enzymatic degradation (see the chemical structure in ). It has been reported that devices of PLGA degrade in a heterogeneous manner. 3 A model describing this process and based on work by Vert and co-workers was proposed by Park, 4 in which the degradation proceeds more rapidly in the center than at the exterior. This phenomenon is thought to be caused by autocatalytic action of the carboxylic acid end groups of the degrading material trapped in the internal milieu. It has been questioned whether this model is also valid when dealing with microspheres. 7 A more homogeneous microsphere degradation, has been postulated on the basis of observation of homogeneous degradation of small devices such as thin films. In many cases, implantation of a biomaterial results in a foreign body reaction involving macrophages, which phagocytose small particles of the degrading materials. After phagocytosis by these cells, degradation may continue inside the phagosome, which plays a crucial role in degradation of internalized materials. The in vivo response to implanted biomaterials is in general studied by histology, which examines the tissue reaction by light microscopy. The limitation of these studies is that, although morphological changes can be studied, no information about the chemical composition of the degrading materials can be obtained from such measurements. Raman spectroscopy allows one to study the chemical bonds involved in degradation of these polymers by detecting intensity and wavelength changes in the vibrational bands of these bonds. We studied the degradation of PLGA microspheres, after macrophage phagocytosis in vitro, by nonresonant confocal Raman spectroscopy and imaging (see and 3). The PLGA microspheres (50:50 glycolide/lactide by weight) were produced by a so-called salting-out procedure described before. 9,10 Scanning electron microscopy (SEM) showed that their size varied between 1 and 10 µm in diameter and that they were of solid nature ( ). The microspheres were sterilized in 70% ethanol for 15 min and then washed in sterile PBS. After washing, the material was opsonized for 30 min using human serum. The microspheres were then added to a macrophage cell line (RAW 264.7) cultured on poly-L-lysine-coated CaF 2 slides, in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, and antibiotics. The cells were cultured for 1 and 2 weeks at which point they were fixed using 4% paraformaldehyde for 30 min. The samples were washed and placed in PBS for Raman measurements, which were performed using a home-built confocal Raman microscope as described previously. 11 A pinhole of 15 µm diameter was employed, providing an axial resolution (FWHM) of 1.5 µm. During Raman imaging, we scanned an intracellular area of 7.5 × 7.5 µm 2 , using a signal accumulation time of 1 s per pixel at 100 mW 647.1 nm excitation.

Scaffolds from poly(ethylene oxide) and poly(butylene terephthalate), PEOT/PBT, with a PEO molecular weight of 1,000 and a PEOT content of 70 weight% (1000PEOT70PBT30) were prepared by leaching salt particles (425–500 μm). Scaffolds of 73.5, 80.6 and 85.0% porosity were treated with a CO2 gas plasma and seeded with rat bone marrow stromal cells (BMSCs). After in vitro culture for 7 days (d) in an osteogenic medium the scaffolds were subcutaneously implanted for 4 weeks in nude mice. Poly(d, l-lactide) (PDLLA) and biphasic calcium phosphate (BCP) scaffolds were included as references. After 4 weeks (wks) all scaffolds showed ectopic formation of bone and bone marrow. For the scaffolds of different porosities, no significant differences were observed in the relative amounts of bone (7–9%) and bone marrow (6–11%) formed, even though micro computed tomography (μ-CT) data showed considerable differences in accessible pore volume and surface area. 1000PEOT70PBT30 scaffolds with a porosity of 85% could not maintain their original shape in vivo. Surprisingly, 1000PEOT70PBT30 scaffolds with a porosity of 73.5% showed cartilage formation. This cartilage formation is most likely due to poorly accessible pores in the scaffolds, as was observed in histological sections. μ-CT data showed a considerably smaller accessible pore volume (as a fraction of the total volume) than in 1000PEOT70PBT30 scaffolds of 80.6 and 85.0% porosity. BMSC seeded PDLLA (83.5% porosity) and BCP scaffolds (29% porosity) always showed considerably more bone and bone marrow formation (bone marrow formation is approximately 40%) and less fibrous tissue ingrowth than the 1000PEOT70PBT30 scaffolds. The scaffold material itself can be of great influence. In more hydrophobic and rigid scaffolds like the PDLLA or BCP scaffolds, the accessibility of the pore structure is more likely to be preserved under the prevailing physiological conditions than in the case of hydrophilic 1000PEOT70PBT30 scaffolds. Scaffolds prepared from other PEOT/PBT polymer compositions, might prove to be more suited.

The aim of this study was to evaluate the cytotoxicity and gelation of thermosensitive chitosan-b-glycerol phosphate (GP) solutions, which undergo sol-gel transition around body temperature. Chitosan 0.5-2% (w/v) mixed with GP 5-20% (w/v) solutions all gel at 378C and possess pH around the physiological range. High GP and chitosan concentrations result in faster gelation time. Extracts of all chitosan concentrations mixed with or without 5% (w/v) GP and 2% (w/v) chitosan combined with 10% (w/v) GP demonstrated up to 34% increase in proliferation rate of goat bone marrow derived mesenchymal stem cells when compared with control medium. Extracts from all other chitosan-GP combinations resulted in reduced cell proliferation relative to control medium. Increasing GP content in the gel resulted in a linear increase in the osmolality of the extracts in contact with the gels. The results of this study indicate that chitosan-GP is a biocompatible hydrogel, extracts of which can stimulate mesenchymal stem cell proliferation at certain concentrations. This material is therefore a promising vehicle for cell encapsulation and injectable tissue-engineering applications.

Over the past decade we have demonstrated numerous times that calcium phosphates can be rendered with osteoinductive properties by introducing specific surface microstructures 1 . Since most of these calcium phosphates contained hydroxyapatite, they are either slowly or not resorbable 2 . Resorbability is an often sought after characteristic of calcium phosphates so that they can be gradually replaced by newly formed bone. The objective of this study was to prepare a resorbable surface microstructured tricalcium phosphate (TCP) ceramic and evaluate its osteoinductive property and resorption rate after intramuscular implantation in dogs. This material was then compared to the established and slowly resorbable osteoinductive biphasic calcium phosphate ceramic (BCP).

A current challenge of synthetic bone graft substitute design is to induce bone formation at a similar rate to its biological resorption, matching bone's intrinsic osteoinductivity and capacity for remodelling. We hypothesise that both osteoinduction and resorption can be achieved by altering surface microstructure of beta-tricalcium phosphate (TCP). To test this, two TCP ceramics are engineered with equivalent chemistry and macrostructure but with either submicron- or micron-scale surface architecture. In vitro, submicron-scale surface architecture differentiates larger, more active osteoclasts--a cell type shown to be important for both TCP resorption and osteogenesis--and enhances their secretion of osteogenic factors to induce osteoblast differentiation of human mesenchymal stem cells. In an intramuscular model, submicrostructured TCP forms 20 % bone in the free space, is resorbed by 24 %, and is densely populated by multinucleated osteoclast-like cells after 12 weeks; howev...

Osteoinductive calcium phosphate (CaP) ceramics can be combined with polymeric carriers to make shapeable bone substitutes as an alternative to autologous bone; however, carriers containing water may degrade the ceramic surface microstructure, which is crucial to bone formation. In this study five novel tricalcium phosphate (TCP) formulations were designed from water-free polymeric binders and osteoinductive TCP granules of different particle sizes (500-1000 lm for moldable putty forms, and 150-500 lm for flowable paste forms). The performance of these novel TCP formulations was studied and compared with control TCP granules alone (both 150-500 and 500-1000 lm). In vitro the five TCP formulations were characterized by their carrier dissolution times and TCP mineralization kinetic profiles in simulated body fluid. In vivo the formulations were implanted in the dorsal muscle and a unicortical femoral defect (Ø = 5 mm) of dogs for 12 weeks. The TCP formulation based on a xanthan gum-glycerol carrier exhibited fast carrier dissolution (1 h) and TCP mineralization (7 days) in vitro, but induced inflammation and showed little ectopic bone formation. This carrier chemistry was thus found to disrupt the early cellular response related to osteoinduction by microstructured TCP. TCP formulations based on carboxymethyl cellulose-glycerol and Polyoxyl 15-hydroxystearate-Pluronic Ò F127 allowed the in vitro surface mineralization of TCP by day 7 and produced the highest level of orthotopic bone bridging and ectopic bone formation, which was equivalent to the control. These results demonstrate that water-free carriers can preserve the chemistry, microstructure, and performance of osteoinductive CaP ceramics.