Development of a biomimetic collagen-hydroxyapatite scaffold for bone tissue engineering using a SBF immersion technique.

The objective of this study was to develop a biomimetic, highly porous collagen-hydroxyapatite (HA) composite scaffold for bone tissue engineering (TE), combining the biological performance and the high porosity of a collagen scaffold with the high mechanical stiffness of a HA scaffold. Pure collagen scaffolds were produced using a lyophilization process and immersed in simulated body fluid (SBF) to provide a biomimetic coating. Pure collagen scaffolds served as a control. The mechanical, material, and structural properties of the scaffolds were analyzed and the biological performance of the scaffolds was evaluated by monitoring the cellular metabolic activity and cell number at 1, 2, and 7 days post seeding. The SBF-treated scaffolds exhibited a significantly increased stiffness compared to the pure collagen group (4-fold increase), while a highly interconnected structure (95%) was retained. FTIR indicated that the SBF coating exhibited similar characteristics to pure HA. Micro-CT showed a homogeneous distribution of HA. Scanning electron microscopy also indicated a mineralization of the collagen combined with a precipitation of HA onto the collagen. The excellent biological performance of the collagen scaffolds was maintained in the collagen-HA scaffolds as demonstrated from cellular metabolic activity and total cell number. This investigation has successfully developed a biomimetic collagen-HA composite scaffold. An increase in the mechanical properties combined with an excellent biological performance in vitro was observed, indicating the high potential of the scaffold for bone TE.

Characterization of esterified hyaluronan-gelatin polymer composites suitable for chondrogenic differentiation of mesenchymal stem cells.

Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, for example, scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation, and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p< 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin-containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, based on which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of alpha5beta1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation.

Biomechanical characterisation of osteosyntheses for proximal femur fractures: helical blade versus screw.

Proximal femur fractures are of main concern for elderly and especially osteoporotic patients. Despite advanced implant modifications and surgical techniques, serious mechanical complication rates between 4-18% are found in conventional osteosyntheses of proximal femur fractures. Clinical complications such as the rotation of the femoral head and the cut-out phenomenon of the fracture fixation bolt are often diagnosed during post-operative treatments. Therefore, efforts in new intramedulary techniques focus on the load bearing characteristics of the implant by developing new geometries to improve the implant-tissue interface. The objective of this investigation was to analyse the osteosynthesis/femur head interaction of two commonly used osteosyntheses, one with a helical blade and the other one with a screw design under different loading conditions. For the comparative investigation the helical blade of the Proximal Femur Nail Antirotation was investigated versus the screw system of the Dynamic Hip Screw. After implantation in a femoral head the loads for rotational overwinding of the implants were analysed. Pull-out forces with suppressed rotation were investigated with analysis of the influence of the previous overwinding. All investigations were performed on human femoral heads taken of patients with average age of 70.3+/-11.8. The bone mineral densities of the human specimens were detected by QCT-scans (average BMD: 338.9+/- 61.3$\frac[\mathit[mg]][\mathit[cm];[3]]$) Prior to cadaveric testing the experimental set-up was validated and special influences were analysed by the use of synthetic foam blocks (Sawbone). The helical blade showed a significant higher torque for the rotation of the femoral head compared to the screw system. The pull-out forces of the blade were substantially lower than of the comparative screw. Taken together the helical blade showed a higher potential of rotational stability, but after a rotation the lower pull-out forces demonstrate a higher degree of damage to the femoral head.

How do anisotropy and age affect fatigue and damage in cancellous bone?

The fatigue behaviour of materials is of particular interest for the failure prediction of materials and structures exposed to cyclic loading. For trabecular bone structures only a few sets of lifetime data have been reported in the literature and structural measures are commonly not considered. The influence of load contributions not aligned with the main physiological axis remains unclear. Furthermore age effects on the fatigue behaviour are not well described. In the present study, different groups of human vertebral cancellous bone were exposed to cyclic compression. The inital modulus and therefore lifetimes were found to be highly dependent on age. The decrease in both with increasing age was much more pronounced in specimens which were not aligned with the main physiological axis. This implies that old bone is much more sensitive to (cyclic) failure loads in general but particularly to loads which are not coincident with the physiological main axis.

Influence of pore size on tensile strength, permeability and porosity of hyaluronan-collagen scaffolds.

Recent investigations have shown the importance of scaffold pore size on the realisation of tissue engineered cartilage which promotes cell adhesion, proliferation and differentiation. The objective of this study was to investigate the influence of pore size on the mechanical properties, the permeability and the porosity of hyaluronan-collagen scaffolds. Hyaluronan-collagen scaffolds with three different mean pore sizes (302.5, 402.5 and 525 microm) have been produced according to a standardised protocol. The maximum stress at rupture, the Young's Moduli, permeability and porosity of the scaffolds were investigated. The permeability was determined both empirically and mathematically. Increased pore sizes indicated a larger stress at rupture as well as increased Young's Moduli. Porosity and permeability were raised by increasing pore sizes. The mathematically calculated permeability showed the same trend. The results indicate a higher mechanical stability for scaffolds with larger pores. The experimental and mathematical experiments both show increased permeability and fluid mobility for larger pores in scaffolds. Morphological changes resulting from the alteration of pore size led to non-correlation between the calculated and the experimental permeability.

Mechanical characteristics of articular cartilage bonds.

BACKGROUND: Sutures for adaptation of articular cartilage are used in arthritis therapy techniques. However, little is known about the mechanical functionality of these sutures. The objective of the present work was to compare the mechanical properties of articular cartilage bonds either generated by suture, or, alternatively, by chemical cross-linking of the opposing surfaces or in vitro integrative repair of cartilage blocks. METHODS: Bonding was achieved by suture in varying numbers, positions and orientations, by surface cross-linking using carbodiimide in combination with pepsin or guanidine (immediate bonding), or by cultivation for 14 days, either with or without testosterone. The mechanical properties of the cartilage bonds were measured under tensile loading. FINDINGS: Suture led to the highest maximal load at failure and by far to the highest strain and lowest stiffness of the bonded samples. Immediate bonding by chemical cross-linking in combination with pepsin led to a low force at failure, but the highest stiffness, as compared to all other groups. Cultivation in the presence of testosterone led to a higher force at failure and a higher strain than chemical cross-linking. INTERPRETATION: Suture technique for bonding of cartilage surfaces leads to a very elastic adaptation which allows synovial fluid flow in between the interface of cartilage wounds. Long-term bonding of cartilage wounds would be counteracted by a fluid flow through the interface during motion of the joint. Immediate bonding of cartilage wounds by chemical cross-linking reagents might be a useful alternative tool. Even more promising, with regard to the mechanical properties, appears to be integrative repair of cartilage blocks stimulated by testosterone.

Bonding of articular cartilage using a combination of biochemical degradation and surface cross-linking.

After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair.

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin-1beta.

OBJECTIVE: Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin-1beta (IL-1beta), and combinations thereof. METHODS: Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17beta-estradiol, and dehydroepiandrosterone (DHEA), with or without IL-1beta. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix. RESULTS: Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 microg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration-dependent stimulation of integration (with maximum values observed with DHEA at 3 x 10(-5)M, testosterone at 10(-8)M, and 17beta-estradiol at 10(-11)M). For testosterone and 17beta-estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL-1beta at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL-1beta and sex hormones, integration of articular cartilage was reduced. CONCLUSION: This is the first study to demonstrate that steroid hormones such as 17beta-estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL-1beta. In the absence of IL-1beta or after neutralization of IL-1beta, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.