Klaas de Groot

Tissue engineering of bone

Bone is a dynamic, highly vascularized tissue with the unique capacity to heal and to remodel depending on line of stress (Buckwalter et al, 1995ab). It exhibits the unlikely combination of high compressive strength and tensile strength due to the composite of calcium phosphate salts (hydroxyapatite) and collagen, respectively (Yaszemski et al, 1996a). It is difficult to find materials to mimic such a complex system when filling bone defects. However, current research capitalizes on the dynamic properties of bone by providing a biodegradable scaffold to guide healing.

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.

Proliferation and differentiation of osteoblast-like MC3T3-E1 cells on biomimetically and electrolytically deposited calcium phosphate coatings

Biomimetic and electrolytic deposition are versatile methods to prepare calcium phosphate coatings. In this article, we compared the effects of biomimetically deposited octacalcium phosphate and carbonate apatite coatings as well as electrolytically deposited carbonate apatite coating on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. It was found that MC3T3-E1 cells cultured on the biomimetically deposited carbonate apatite coating demonstrated the greatest proliferation rate and the highest differentiation potential. Cells on the biomimetically deposited octacalcium phosphate coating had lower proliferation rate before day 7, but higher after that, than those on the electrolytically deposited carbonate apatite coating. There was no difference on the expression of early differentiation markers, that is, alkaline phosphatase activity and collagen content, between biomimetically deposited octacalcium phosphate and electrolytically deposited carbonate apatite coatings. However, higher expression of late differentiation markers, that is, osteocalcin and bone sialoprotein mRNA, was found on the biomimetically deposited octacalcium phosphate coating on day 14. These results suggest that the difference in in vitro osteoblast cell performance of calcium phosphate coatings might relate to their physicochemical properties. Biomimetic carbonate apatite coating is the most favorable surface for the proliferation and differentiation of MC3T3-E1 cells.

Electrolytic deposition of lithium into calcium phosphate coatings

OBJECTIVESnLithium ions stimulate the Wnt signaling pathway and the authors previously demonstrated that lithium enhances the proliferation of tissue cultured human mesenchymal stem cells. The aim of this study was to prepare and characterize a calcium phosphate/lithium coating by means of electrolytic deposition. It was hypothesized that the hybrid coatings would enhance the proliferation of MG63 osteoblast-like cells in vitro.nnnMETHODSnCalcium phosphate coatings were electrolytically deposited in electrolytes containing 0, 0.5 and 5g/L lithium chloride, respectively. They were characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The coating thickness, lithium content and release profile were also measured. The cell attachment and cell-doubling index of MG63 cells on these coatings were determined through a Cell Counting Kit-8.nnnRESULTSnLithium inhibited calcium phosphate deposition in a dose-dependent manner. Both crystallinity and thickness of the coatings were reduced with increasing lithium concentration in the electrolyte. The incorporation of lithium was 2.2 and 5.5microg/mg, respectively. The hybrid coatings demonstrated a burst lithium release within half an hour upon immersion into simulated physiological solution. Both attachment and early proliferation of MG63 cells on these hybrid coatings were enhanced.nnnSIGNIFICANCEnThese results suggest that lithium can be effectively incorporated into calcium phosphate coatings. The incorporation of lithium interferes with calcium phosphate deposition; however, it enhances the biocompatibility of the coatings.

Predictive value of in vitro and in vivo assays in bone and cartilage repair : What do they really tell us about the clinical performance?

The continuous increase of life expectancy leads to an expanding demand for repair and replacement of damaged and degraded organs and tissues. Recent completion of a first version of the human genome sequence is a great breakthrough for the field of pharmaceutics. It is conceivable that new developments in pharmaceutical research will result in a large number of novel and improved medicines. A similar development is expected in the field of biomaterials designed for bone and cartilage repair and replacement. Spinal fusions and repairs of bone defects caused by trauma, tumors, infections, biochemical disorders and abnormal skeletal development, are some examples of the frequently performed surgeries in the clinic. For most of these surgeries, there is a great need for bone graft substitutes. Similarly, the number of patients worldwide experiencing joint pain and loss of mobility through trauma or degenerative cartilage conditions is considerable, and yet, few approaches employed clinically are capable of restoring long-term function to damaged articular cartilage1, 2. Therefore, new materials and techniques need to be developed.

Laryngotracheal reconstruction with porous titanium in rabbits: are vascular carriers and mucosal grafts really necessary?

Laryngotracheal reconstruction requires a supportive structure with a mucosal lining, which needs a vascular supply in order to regenerate properly. We investigated the necessity of a vascular carrier and mucosal graft when using porous titanium for laryngotracheal reconstruction. Surgical defects of the laryngotracheal complex in 22 rabbits were reconstructed with: (a) porous titanium implanted on a vascularized fascia combined with a buccal mucosal graft (first stage) before transposing to the neck area (second stage); (b) porous titanium implanted on a vascularized fascia (first stage) combined with a mucosal graft (second stage); (c) porous titanium on a pedicled fascia flap; and (d) porous titanium alone. The grafts were tolerated well. Re‐epithelialization occurred in all groups. Normal mucosa with a submucosal layer containing vital cells was noted using the titanium implants. Blood vessels were grown in the pores of the titanium scaffold to supply the overlying mucosa. The scaffold was well integrated in the adjacent tracheal cartilage and surrounding tissues, except in the two cases that showed titanium displacement. Inflammation and granulation formation were seen in most rabbits in groups III and IV, initiated probably by the use of buccal mucosal grafts. Reconstruction of a rabbits trachea using composites of porous titanium, mucosal grafts and a fascia flap is feasible. Titanium seems to meet the requirements needed for closing a small defect of the tracheal wall and allows for re‐epithelialization. For larger defects, a vascular carrier with a mucosal graft is probably indispensable to ensure the process of re‐epithelialization. Copyright

Screening molluscan cDNA expression libraries with anti-shell matrix antibodies

In a previous paper [Marin et al., Protein Expr. Purif. 23 (2001) 175], we showed that polyclonal antibodies raised against molluscan shell matrices could be useful tools for visualizing shell proteins after a preparative fractionation of the shell matrix. In this paper, we have used the same antibodies for screening a cDNA library constructed from mantle tissues of the nacro-prismatic bivalve Pinna nobilis. The immunoscreening led to the identification of a new protein, mucoperlin [Marin et al., J. Biol. Chem. 275 (2000) 20667], which was subsequently overexpressed. A polyclonal antibody was obtained from the recombinant mucoperlin. In a control assay, we unambiguously demonstrated that this antibody and one of the sera used for the initial screening hybridize with the same clones. We assess that screening cDNA libraries with antibodies elicited against unfractionated calcifying matrices is a good alternative to oligonucleotide screening techniques, particularly in the field of molluscan biomineralization where only few gene sequences are known.