C.A. van Blitterswijk

Injectable chitosan-based hydrogels for cartilage tissue engineering

Water-soluble chitosan derivatives, chitosan-graft-glycolic acid (GA) and phloretic acid (PA) (CH-GA/PA), were designed to obtain biodegradable injectable chitosan hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and H2O2. CH-GA/PA polymers were synthesized by first conjugating glycolic acid (GA) to native chitosan to render the polymer soluble at pH 7.4, and subsequent modification with phloretic acid (PA). The CH-GA43/PA10 with a degree of substitution (DS, defined as the number of substituted NH2 groups per 100 glucopyranose rings of chitosan) of GA of 43 and DS of PA of 10 showed a good solubility at pH values up to 10. Short gelation times (e.g. 10 s at a polymer concentration of 3 wt%), as recorded by the vial tilting method, were observed for the CH-GA43/PA10 hydrogels using HRP and H2O2. It was shown that these hydrogels can be readily degraded by lysozyme. In vitro culturing of chondrocytes in CH-GA43/PA10 hydrogels revealed that after 2 weeks the cells were viable and retained their round shape. These features indicate that CH-GA/PA hydrogels are promising as an artificial extracellular matrix for cartilage tissue engineering.

Influence of ionic strength and carbonate on the Ca-P coating formation from SBF×5 solution

Biomimetic calcium-phosphate (Ca-P) coatings were applied on Ti6Al4V by using simulated body fluids concentrated by a factor 5 (SBF×5). The production of SBF×5 solution was possible by decreasing the pH of the solution to approximately 6 using CO2 gas. The subsequent release of this mildly acidic gas led to a pH rise and thus, increasing supersaturation. After immersion for 5 1/2 h a Ca-P coating on Ti6Al4V plates and a precipitate simultaneously formed at pH=6.8. Sodium chloride (NaCl) and hydrogencarbonate (HCO3−) contents were studied in relation to CO2 release and coating formation by changing their individual concentration in SBF×5 solution. On one hand, NaCl-free or low NaCl-content SBF×5 solution led to the earlier aspecific precipitation in the solution than for SBF×5 solution. In contrast, Ca-P coating was formed later and was thinner than the coating obtained in regular SBF×5 solution. High ionic strength delayed precipitation and favored Ca-P heterogeneous nucleation on Ti6Al4V. On the other hand, HCO3− content increased the pH of the solution due to its buffering capacity and influenced the release rate of dissolved CO2. Thus, HCO3− content strongly affected the supersaturation and Ca-P structure. Furthermore, HCO3− favored the attachment of Ca-P mineral on Ti6Al4V by decreasing Ca-P crystal size resulting in a better physical attachment of Ca-P coating on Ti6Al4V substrate.

Nucleation of biomimetic Ca–P coatings on Ti6Al4V from a SBF×5 solution: influence of magnesium

Biomimetic Calcium-Phosphate (Ca-P) coatings were applied by using 5 times concentrated Simulated Body Fluid (SBF x 5) using Carbon Dioxide gas. This process allows the deposition of a uniform Ca-P coating within 24 h. A previous study of our process emphasized the importance of hydrogenocarbonate ions (HCO-3), a crystal growth inhibitor. The aim of the present study was to investigate the role of the other crystal growth inhibitor present in SBF x 5, Magnesium (Mg2+), on the Ca-P coating formation. Several SBF x 5 solutions were prepared with various Mg2+ and HCO3 contents. No Ca-P deposits were detected on Ti6A14V substrate soaked for 24h in a Mg-free SBF x 5 solution, whereas by increasing HCO-3 content in a Mg-free SBF x 5 solution, a Ca-P coating developed on Ti6A14V substrate. Therefore, it appeared that Mg2+ has a stronger inhibitory effect on apatite crystal growth than HCO-3. Nevertheless, Mg2+ plays also another important role as suggested by depth profile X-ray Photoelectron Spectroscopy (XPS) of the Ca-P coating obtained from SBF x 5 solution. Ca2+ and Mg2+ contents increased significantly at the titanium/coating interface. Therefore, Ca2+ and Mg2+ initiated Ca-P coating from SBF x 5 solution. The relatively high interfacial concentration in Mg2+ favors heterogeneous nucleation of tiny Ca-P globules onto the substrate. So physical adhesion is enhanced at the early stage of the coating formation.

Biomimetic calcium phosphate coatings on Ti6Al4V: a crystal growth study of octacalcium phosphate and inhibition by Mg2+ and HCO3−

The biomimetic approach for coating metal implants allows the deposition of new calcium phosphate (Ca-P) phases. Films elaborated at physiological conditions might have structures closer to bone mineral than hydroxylapatite (HA) plasma-sprayed coatings. In this study, different Ca-P coatings have been deposited through a two-step procedure. After cleaning and etching, Ti6Al4V plates were pretreated by soaking in a simulated body fluid (SBF), i.e., a solution containing inorganic components in concentration more or less similar to body fluids: a thin amorphous carbonated Ca-P layer precipitated on the metal substrate. Second, by soaking these thinly coated metal substrates in another SBF, with different concentrations, the thin amorphous carbonated Ca-P layer led to the fast precipitation of a second and thick Ca-P layer. Different SBF solutions were used in order to investigate the influence of magnesium and carbonate ions. From SBF containing only Ca2+ and HPO4(2-) ions, an octacalcium phosphate layer grew epitaxially on the substrate. When Mg2+ was added into this SBF, the coating was composed of Ca-deficient apatite crystals, while the addition of HCO3- in SBF led to the formation of a B-carbonated apatite layer. Magnesium and carbonate acted as inhibitors of crystal growth. The three phases obtained by our biomimetic process are closer to bone mineral structure than plasma-sprayed HA. Therefore, the obtained results may be particularly relevant for the development of biomimetic Ca-P coatings with optimal bioactivity.

Effect of fibroblasts on epidermal regeneration.

Summary Background There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies.

Bone Tissue-Engineered Implants Using Human Bone Marrow Stromal Cells: Effect of Culture Conditions and Donor Age

At present, it is well known that populations of human bone marrow stromal cells (HBMSCs) can differentiate into osteoblasts and produce bone. However, the amount of cells with osteogenic potential that is ultimately obtained will still be dependent on both patient physiological status and culture system. In addition, to use a cell therapy approach in orthopedics, large cell numbers will be required and, as a result, knowledge of the factors affecting the growth kinetics of these cells is needed. In the present study we investigated the effect of dexamethasone stimulation on the in vivo osteogenic potential of HBMSCs. After a proliferation step, the cells were seeded and cultured on porous calcium phosphate scaffolds for 1 week, and then subcutaneously implanted in nude mice for 6 weeks, in order to evaluate their in vivo bone-forming ability. Furthermore, the effect of donor age on the proliferation rate of the cultures and their ability to induce in vivo bone formation was studied. In 67% of the assayed patients (8 of 12), the presence of dexamethasone in culture was not required to obtain in vivo bone tissue formation. However, in cultures without bone-forming ability or with a low degree of osteogenesis, dexamethasone increased the bone-forming capacity of the cells. During cellular proliferation, a significant age-related decrease was observed in the growth rate of cells from donors older than 50 years as compared with younger donors. With regard to the effect of donor age on in vivo bone formation, HBMSCs from several donors in all age groups proved to possess in vivo osteogenic potential, indicating that the use of cell therapy in the repair of bone defects can be applicable irrespective of patient age. However, the increase in donor age significantly decreased the frequency of cases in which bone formation was observed.

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

A comparison of the osteoinductive potential of two calcium phosphate ceramics implanted intramuscularly in goats.

The osteoinductive potential, or bone induction potency, of two calcium phosphate ceramics was evaluated after intramuscular implantation in goats. The ceramics were comprised of hydroxyapatite (HA) and biphasic calcium phosphate (BCP), the later of which contained a 85/15 mixture of hydroxyapatite and tricalcium phosphate (TCP). Both ceramics had a similar macroporosity of around 55% and a pore distribution between 100 and 800 μm. Besides the difference in chemistry, BCP was also microporous and hence had a different surface microstructure. After implantation in the back muscles of four goats for 12 weeks, all 8 BCP samples (7×7×7 mm3) showed the presence of bone formation in the macropores (1±1%), while no bone was found in any of the HA samples. The used BCP can therefore be characterized as an osteoinductive material. Having the ability to induce bone formation in soft tissues, the BCP presented herein may be a useful biomaterial for bone repair when combined with cultured osteogenic cells, growth factors or both.

Macropore tissue ingrowth: a quantitative and qualitative study on hydroxyapatite ceramic

The aim of this study was to obtain more information about macropore tissue ingrowth into the pores of sintered hydroxyapatite implanted in the rat middle ear, for the assessment of the usefulness of this material in reconstructive middle-ear surgery. The exudate filing the pores during the early post-operative period was gradually replaced by equal amounts of fibrous tissue and bone. The percentage of the macropore area occupied by bone was directly correlated with the macropore size. Bone was deposited not only from the pore wall towards the pore centre, but also in the opposite direction. Bonding osteogenesis was demonstrated. At sites of mechanical irritation, the presence of multinucleated cells and proliferatively active mononuclear phagocytes persisted for as long as a year. Under appropriate conditions hydroxyapatite seems to be a promising material for bone substitution in reconstructive middle-ear surgery.

Bioreactions at the tissue/ hydroxyapatite interface

The events at the hydroxyapatite implant material/tissue interface in the rat middle ear were studied by light microscopy, autoradiography, morphometry, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray microanalysis. Deposition of calcium, partially in the form of calcium phosphate, was found at the interface. Resorption of the implant material occurred as the result of mono- and multinuclear phagocyte activity. Resorption decreased 6 mnth after the operation, possibly due to the decreasing number of phagocytes at the interface and the increasing amount of bone in the macropores.