Jérôme Sohier

Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering.

High strength porous scaffolds and mesenchymal stem cells are required for bone tissue engineering applications. Porous titanium scaffolds (TiS) with a regular array of interconnected pores of 1000 microm in diameter and a porosity of 50% were produced using a rapid prototyping technique. A calcium phosphate (CaP) coating was applied to these titanium (Ti) scaffolds with an electrodeposition method. Raman spectroscopy and energy dispersive X-ray analysis showed that the coating consisted of carbonated hydroxyapatite. Cross-sectioned observations by scanning electron microscopy indicated that the coating evenly covered the entire structure with a thickness of approximately 25 microm. The bonding strength of the coating to the substrate was evaluated to be around 25 MPa. Rat bone marrow cells (RBMC) were seeded and cultured on the Ti scaffolds with or without coating. The Alamar Blue assay provided a low initial cell attachment (40%) and cell numbers were similar on both the uncoated and coated Ti scaffolds after 3 days. The Ti scaffolds were subsequently implanted subcutaneously for 4 weeks in syngenic rats. Histology revealed the presence of a mineralized collagen tissue in contact with the implants, but no bone formation. This study demonstrated that porous Ti scaffolds with high strength and defined geometry may be evenly coated with CaP layers and cultured mesenchymal stem cells for bone tissue engineering.

The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties.

We previously identified multipotent stem cells within the lamina propria of the human olfactory mucosa, located in the nasal cavity. We also demonstrated that this cell type differentiates into neural cells and improves locomotor behavior after transplantation in a rat model of Parkinsons disease. Yet, next to nothing is known about their specific stemness characteristics. We therefore devised a study aiming to compare olfactory lamina propria stem cells from 4 individuals to bone marrow mesenchymal stem cells from 4 age- and gender-matched individuals. Using pangenomic microarrays and immunostaining with 34 cell surface marker antibodies, we show here that olfactory stem cells are closely related to bone marrow stem cells. However, olfactory stem cells also exhibit singular traits. By means of techniques such as proliferation assay, cDNA microarrays, RT-PCR, in vitro and in vivo differentiation, we report that when compared to bone marrow stem cells, olfactory stem cells display (1) a high proliferation rate; (2) a propensity to differentiate into osseous cells; and (3) a disinclination to give rise to chondrocytes and adipocytes. Since peripheral olfactory stem cells originate from a neural crest-derived tissue and, as shown here, exhibit an increased expression of neural cell-related genes, we propose to name them olfactory ectomesenchymal stem cells (OE-MSC). Further studies are now required to corroborate the therapeutic potential of OE-MSCs in animal models of bone and brain diseases.

A novel method to obtain protein release from porous polymer scaffolds: emulsion coating.

To obtain the controlled release of proteins from macro-porous polymeric scaffolds, a novel emulsion-coating method has been developed. In this process, a water-in-oil emulsion, from an aqueous protein solution and a polymer solution, is forced through a prefabricated scaffold by applying a vacuum. After solvent evaporation, a polymer film, containing the protein, is then deposited on the porous scaffold surface. This paper reports the effect of processing parameters on the emulsion coating characteristics, scaffold structure, and protein release and stability. Poly(ether-ester) multiblock copolymers were chosen as the polymer matrix for both scaffolds and coating. Macro-porous scaffolds, with a porosity of 77 vol% and pores of approximately 500 microm were prepared by compression moulding/salt leaching. A micro-porous, homogeneous protein-loaded coating could be obtained on the scaffold surface. Due to the coating, the scaffold porosity was decreased, whereas the pore interconnection was increased. A model protein (lysozyme) could effectively be released in a controlled fashion from the scaffolds. Complete lysozyme release could be achieved within 3 days up to more than 2 months by adjusting the coated emulsion parameters. In addition, the coating process did not reduce the enzymatic activity. This new method appears to be promising for tissue engineering applications.

Porous beta tricalcium phosphate scaffolds used as a BMP-2 delivery system for bone tissue engineering

Macroporous beta tricalcium phosphate (beta-TCP) scaffolds were evaluated as potential carriers and delivery systems for bone morphogenetic protein-2 (BMP-2). Chemical etching was performed to increase the available surface and thus the protein loading. X-ray diffraction and infrared spectrocopy analyses confirmed the preparation of pure beta-TCP scaffolds. Scanning electron microscopy revealed interconnected porosity (64%) and a microporous surface after chemical etching. Scaffolds loaded with 30 and 15 microg of BMP-2 were implanted respectively into the back muscles and into femoral defects (condyle and diaphysis) of rabbits for 4 weeks. Histological observations confirmed the activity of the BMP-2 released from the scaffolds. Intramuscularly, bone was formed within the BMP-2-loaded scaffold pores. In the bone defects, the effect of released BMP-2 was similarly noticeable, as evaluated by histomorphometry. The incorporation of BMP-2 resulted in an amount of newly formed bone that was 1.3 times higher than with unloaded scaffolds. The implant site, however, did not have an effect on bone formation as no statistical differences were measured between cortical (diaphysis) and trabecular (condyle) defects. These results indicate the suitability of chemically etched beta-TCP scaffolds as BMP-2 carriers, in the context of bone regeneration.

Critical factors in the design of growth factor releasing scaffolds for cartilage tissue engineering

Background: Trauma or degenerative diseases of the joints are common clinical problems resulting in high morbidity. Although various orthopedic treatments have been developed and evaluated, the low repair capacities of articular cartilage renders functional results unsatisfactory in the long term. Over the last decade, a different approach (tissue engineering) has emerged that aims not only to repair impaired cartilage, but also to fully regenerate it, by combining cells, biomaterials mimicking extracellular matrix (scaffolds) and regulatory signals. The latter is of high importance as growth factors have the potency to induce, support or enhance the growth and differentiation of various cell types towards the chondrogenic lineage. Therefore, the controlled release of different growth factors from scaffolds appears to have great potential to orchestrate tissue repair effectively. Objective: This review aims to highlight considerations and limitations of the design, materials and processing methods available to create scaffolds, in relation to the suitability to incorporate and release growth factors in a safe and defined manner. Furthermore, the current state of the art of signalling molecules release from scaffolds and the impact on cartilage regeneration in vitro and in vivo is reported and critically discussed. Methods: The strict aspects of biomaterials, scaffolds and growth factor release from scaffolds for cartilage tissue engineering applications are considered. Conclusion: Engineering defined scaffolds that deliver growth factors in a controlled way is a task seldom attained. If growth factor delivery appears to be beneficial overall, the optimal delivery conditions for cartilage reconstruction should be more thoroughly investigated.

Hydrogel/calcium phosphate composites require specific properties for three-dimensional culture of human bone mesenchymal cells.

To provide multipotent cells with a three-dimensional environment closer to bone matrix, an engineered construct mimicking bone components has been designed and evaluated. A biocompatible hydrogel (silated hydroxypropylmethyl cellulose) was used as an extra-cellular matrix while biphasic calcium phosphate ceramic particles were used to replace mineralized matrix. Finally, human bone mesenchymal cells were cultured in three dimensions in the resulting constructs to study their cell viability, proliferation, interactions within the composites, and maintenance of their osteogenic potential. This approach resulted in homogeneous structures in which cells were viable and retained their osteoblastic differentiation potential. However, the cells did not proliferate nor colonize the constructs, possibly because of a lack of suitable interactions with their micro-environment.

Osteoblastic differentiation and potent osteogenicity of three-dimensional hBMSC-BCP particle constructs

Bone tissue engineering usually consists of associating osteoprogenitor cells and macroporous scaffolds. This study investigated the in vitro osteoblastic differentiation and resulting in vivo bone formation induced by a different approach that uses particles as substrate for human bone marrow stromal cells (hBMSCs), in order to provide cells with a higher degree of freedom and allow them to synthesize a three‐dimensional (3D) environment. Biphasic calcium phosphate (BCP) particles (35 mg, ~175 µm in diameter) were therefore associated with 4 × 105 hBMSCs. To discriminate the roles of BCP properties and cell‐synthesized 3D environments, inert glass beads (GBs) of similar size were used under the same conditions. In both cases, high cell proliferation and extensive extracellular matrix (ECM) production resulted in the rapid formation of thick cell‐synthesized 3D constructs. In vitro, spontaneous osteoblastic differentiation was observed in the 3D constructs at the mRNA and protein levels by monitoring the expression of Runx2, BMP2, ColI, BSP and OCN. The hBMSC–BCP particle constructs implanted in the subcutis of nude mice induced abundant ectopic bone formation after 8 weeks (~35%, n = 5/5). In comparison, only fibrous tissue without bone was observed in the implanted hBMSC–GB constructs (n = 0/5). Furthermore, little bone formation (~3%, n = 5/5) was found in hBMSC–macroporous BCP discs (diameter 8 × 3 mm). This study underlines the lack of correspondence between bone formation and in vitro differentiation assays. Furthermore, these results highlight the importance of using BCP as well as a 3D environment for achieving high bone yield of interest for bone engineering. Copyright

Release of small water-soluble drugs from multiblock copolymer microspheres: a feasibility study

Poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) multiblock copolymer was investigated as a possible matrix for controlled delivery of small water-soluble drugs. Two molecules were selected as sustained release candidates from microspheres: leuprorelin acetate (peptide of Mw = 1270 D) and vitamin B(12) (Mw = 1355 D). First, vitamin B(12)-loaded microspheres were prepared using a double emulsion method and preparation parameters were varied (surfactant in the first emulsion and copolymer composition). The resulting microsphere structure, entrapment efficiency and release rate were evaluated. Vitamin B(12)-loaded microsphere parameters could easily be tailored to achieve specific requirements. The addition of surfactant in the first preparation process led to a significant increase of the microsphere entrapment efficiency, whereas the decrease of the PEGT copolymer content allowed the release rates from microspheres to be precisely decreased. However, leuprorelin acetate-loaded microspheres did not show the same characteristics when prepared with the same parameters, possibly because of a high water solubility discrepancy between the vitamin B(12) and the peptide. This study shows the suitability of PEGT/PBT microspheres as a controlled release system for vitamin B(12), but not for leuprorelin acetate. It also underlines the necessity of tailored development for each individual drug and emphasizes the risk of using model molecules.

Novel and simple alternative to create nanofibrillar matrices of interest for tissue engineering.

Synthetic analogs to natural extracellular matrix (ECM) at the nanometer level are of great potential for regenerative medicine. This study introduces a novel and simple method to produce polymer nanofibers and evaluates the properties of the resulting structures, as well as their suitability to support cells and their potential interest for bone and vascular applications. The devised approach diffracts a polymer solution by means of a spraying apparatus and of an airstream as sole driving force. The resulting nanofibers were produced in an effective fashion and a factorial design allowed isolating the processing parameters that control nanofiber size and distribution. The nanofibrillar matrices revealed to be of very high porosity and were effectively colonized by human bone marrow mesenchymal cells, while allowing ECM production and osteoblastic differentiation. In vivo, the matrices provided support for new bone formation and provided a good patency as small diameter vessel grafts.

Healing of long-bone defects in sheep metatarsals using bioceramics and mesenchymal stem cells

Background Autologous bone is the most effective bone graft procedure but is limited in quantity and requires a second surgery site. These drawbacks have encouraged scientists and surgeons to find alternative approaches. The aim of this study was to investigate the ability of biphasic calcium phosphate ceramic granules to promote bone regeneration in a preclinical model. Methods In this work, a 2.5-cm critical-sized bone defect was created using Masquelets technique in sheep metatarsals. The defect was filled with a poly-methylmethacrylate spacer, which was replaced 6 weeks later with biphasic calcium phosphate granules, with or without autologous mesenchymal stem cells (n=7 per group). Results After 12 weeks, nondecalcified histology demonstrated good and homogeneous bone formation for biphasic calcium phosphate ceramic granules, with or without autologous mesenchymal stem cells. No significant difference was observed between the two groups; however, all the implants were able to support bone formation and for certain cases exhibited bone unions. Conclusions These results highlighted the potential of biphasic calcium phosphate ceramic granules to be used as scaffolds for mesenchymal stem cells in the reconstruction of critical-sized bone defects instead of autologous bone grafts.