For the generation of constructs a solid carrier (Sponceram HA^®, Zellwerk, Germany) with a diameter of 4.55 mm and a height of 2 mm was used. The carrier consisted of hydroxylapatite, derived by partially sintering ground porcine bone.

In this work, the surface structure of carriers was modified to determine the influence of the topography on cartilage formation in vitro. In a previous study, surface structuring of dense calcium phosphates was done by grinding and ultrasonic milling using abrasive B4C particles [25]. Considering the surface defect formation observed thereby on some of the dense and therefore hard samples, in the present study a much less abrasive procedure was used in order to reduce defect formation on Sponceram HA carriers; e.g. plain paper (smooth celluloses) and foils used for inkjet printing (rough polymer) were used as tools for careful manual grinding. The results confirm that already these rather smooth tools are effective for surface topography tailoring of Sponceram HA carriers.

The surface topography of each carrier was characterized with the aid of a Charge-coupled Device (CCD) Camera (see below). Before cultivation, carriers were placed in phosphate-buffered saline (PBS, Roth, Germany) overnight which was followed by an autoclaving step.

For specification of the surface structure of carriers a microscope (InfiniteFocus, Alicona, Germany) with a Charge-coupled Device Camera was used which generates a xyz data set of the topography. This can be used to create a 3D image by respective post processing. Furthermore, the Alicona system can be used to generate data concerning roughness and/or wear which, however, are only to a certain but limited degree comparable with the well known data obtained by tactile measurements. Therefore, in this study only 3D images are used for evaluating the variation in surface topography as shown in Fig. (2).

Fig. (1). Alicona image of a Sponceram HA carrier as used in this investigation. The defects are most probably due to cracking along some large agglomerates formed during calcinations of the porcine bone (each scale bar = 4,55 mm).

Fig. (2). Alicona images of (1) as received commercial grade Sponceram HA; (2) after surface tailoring using plain paper; (3) ink jet polymer sheet as grinding tools (scale bar = 200 µm).

Fig. (3). SEM pictures of a cell layer grown on modified carrier surfaces (1) carrier without cells; (2) cell layer on HA carrier as received; (3) cell layer on HA carrier grinded using plain paper; (4) cell layer on HA carrier grinded using ink jet polymer sheet foil (scale bar = 20 µm).

Fig. (4). Biomechanical parameters wet weight, height and Young’s Modulus of cartilage-constructs cultured (1) without using a carrier; (2) with a HA carrier as received; (3) with a HA carrier grinded using plain paper; (4) with a HA carrier grinded using ink jet polymer sheet foil (n=6, p<0.05).

Fig. (5). Biochemical parameters glycosaminoglycan concentration, DNA concentration and GAG to DNA ratio of cartilage-constructs cultured (1) without using a carrier; (2) with a HA carrier as received; (3) with a HA carrier grinded using plain paper; (4) with a HA carrier grinded using ink jet polymer sheet foil (n=6, p<0.05).

Fig. (6). Histological staining for glycosaminoglycans with Safranin O (1) constructs cultured without using a carrier; (2) constructs cultured with HA carrier as received; (3) constructs cultured with a HA carrier grinded using plain paper; (4) constructs cultured with a HA carrier grinded using ink jet polymer sheet foil (scale bar = 100 µm).

Fig. (7). Immunohistological staining: left column for collagen type I, right column for collagen type II (1a)/(1b) constructs cultured without using a carrier; (2a)/(2b) constructs cultured with a HA carrier as received; (3a)/(3b) constructs cultured with a HA carrier grinded using plain paper; (4a)/(4b) constructs cultured with a HA carrier grinded using ink jet polymer sheet; (scale bar = 100 µm).

In general, this allows a high resolution characterization (approx. at the best 10 nm in z direction and 1-2 µm in x-y direction) of rough and non-plain surfaces in non contact mode.

Chondrocytes were isolated from a knee joint (femur) of an approximately 5 month old domestic pig by using hyaluronidase type III solution (Sigma-Aldrich, Germany), trypsin (Roth) and collagenase type Ia solution (Sigma-Aldrich).

Cartilage-carrier-constructs were generated according to the concept described above [22, 23]. Starting with an initial cell number of 2x10⁵ cells for the generation of six constructs, chondrocytes were expanded in T-flasks (Roth) until passage 3 (step a). Proliferation of the cells was performed in DMEM (Dulbecco`s Modified Eagle Medium, PAA, Germany) supplemented with 10 % (v/v) fetal calf serum (FCS, PAA), penicillin/streptomycin (100 U mL^-1 penicillin and 100 µg mL^-1 streptomycin, PAA), 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Roth) and 10 ng mL^-1 FGF-b (human recombinant Fibroblast Growth Factor basic, CellConcepts, Germany) [26].

Chondrocytes were trypsinated from the T-flasks and 2x10⁵ chondrocytes were sedimented onto each calcium phosphate carrier (see above) to form a cell layer (step b). To initiate cell proliferation within two weeks of cultivation, the above mentioned medium was used. Simultaneously, chondrocytes from the same preculture were immobilized in alginate beads (1x10⁶ cells per mL alginate) during two weeks for re-differentiation and production of cartilage matrix (step c).

After recovering cells from the gel, cartilage-constructs were prepared. Therefore, 1.8x10⁶ re-differentiated cells were centrifuged onto each cell coated carrier, located in a special device [22]. For the preparation of cartilage-constructs without using any carrier, 1.8x10⁶ cells harvested from alginate culture were centrifuged in tubes (50 mL falcon tubes, Roth). For two days, constructs remained in their devices or tubes to permit aggregation of cells and extracellular matrix. Afterwards, cartilage-constructs (same procedure for both methods) were transferred to 12-well-plates (Roth) and cultivated for three weeks altogether in a high-density cell culture. Eight constructs of each condition were prepared.

During cultivation of alginate beads and cartilage-carrier-constructs, DMEM (PAA) supplemented with 10 % (v/v) porcine serum (PS, Gibco, Germany), penicillin/streptomycin (100 U mL^-1 penicillin and 100 µg mL^-1 streptomycin, PAA), 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Roth), 0.28 mM L-ascorbic acid 2-phosphate and 1 mM cysteine (Sigma-Aldrich) was used to stimulate matrix production. Furthermore, during re-differentiation in alginate gel hIGF-I (100 ng mL^-1, human recombinant Insulin like Growth factor 1, CellConcepts, Germany) and hTGF-β1 (10 ng mL^-1, human recombinant Transforming Growth Factor beta1, CellConcepts) were added to the medium [27].

Medium was exchanged three times a week. All cultivations were performed at 37 °C under an atmosphere of 5 % (v/v) O₂ and 5 % (v/v) CO₂.

To quantify biochemical properties of cartilage constructs, DNA and glycosaminoglycan (GAG) content was determined according to Buschmann et al. [28]. The generated tissue was digested enzymatically with a papain solution (Roche, Germany) overnight. The GAG content was then measured photometrically by staining with 1,9-dimethylmethylene blue chloride (Serva, Germany) with chondroitin sulfate (Sigma-Aldrich) as the standard. After that, DNA content was determined using the fluorescence marker H33258 (Hoechst, Germany) with calf thymus DNA as the standard [for details see 22, 23].

For histological analysis carriers were removed from the constructs after cultivation by the help of tweezers and the generated tissue was fixed in formaldehyde (4 % in PBS, Roth). After dehydrating and embedding in paraffin, 5 µm thick histological sections were prepared with a microtome (Leica, Germany) on object slides (Histo-Bond®, Marienfeldt, Germany). Paraffin was removed by the aid of xylene. Afterwards, histological sections were rehydrated in solutions with decreasing alcohol concentrations.

The formation of collagen type I and collagen type II was confirmed by immunochemical staining of histological sections. In the first step, histological sections were immunostained separately with primary antibodies (Acris Antibodies, Germany) against collagen type I (clone I-8H5) or collagen type II (clone II-4C11). As secondary antibody, a biotinylated antibody (Goat anti-mouse, [IgG (H+L)-biotin], Southern Biotech, USA) was used. Afterwards, these sections were incubated with a streptavidin/alkaline phosphatase complex (Linaris, Germany) and the final color development was carried out with the New Fuchsin chromogen (Sigma-Aldrich) [29]. Nuclei were stained with haematoxilin (Roth).

Distribution of glycosaminoglycans in the cultivated tissue was determined by a Safranin O staining. Therefore, hydrated sections were submerged in Fast Green solution (0.04 % in 0.2 % acetic acid, VWR, Germany), afterwards washed in 1 % acetic acid and then stained with Safranin O (0.2 % in 1 % acetic acid, Sigma-Aldrich).

The biomechanical parameters wet weight (Mettler AE200), height and the Young’s Modulus of the cartilage-constructs were determined. For measuring the height and the Young’s Modulus, a high-precision material testing equipment (Zwicki 1120, Zwick, Germany) was used. The Young’s Modulus was determined by stepwise stress-relaxation tests (five steps with 4 % of the uncompressed cartilage thickness each) according to Korhonen et al. [30]. The criterion for relaxation was a relaxation rate of less than 0.002 N min-1. The Young’s Modulus was calculated from resulting stress-strain curve.

Stability (interface between cartilage and carrier) was established qualitatively by pulling the cartilage carefully with a tweezers.

Scanning electron microscopy (SEM, Leo 1530, Zeiss, Germany) was used to photograph the cell coated carrier after two weeks in culture (step b). At the end of cultivation carriers were kept in a glutaraldehyde solution (5 % in PBS, Sigma-Aldrich) to fix the cells. After a minimum of one day, they were dehydrated by stepwise exchanging water with ethanol (20 %, 40 %, 60 %, 80 %, 100 % ethanol in PBS) and stored in 100 % ethanol. The next day, ethanol was exchanged with amyl acetate (Sigma-Aldrich) for two hours in an amyl acetate-ethanol mixture (1:1 v/v). Afterwards, carriers were stored in 100 % amyl acetate overnight. Before carrying out the SEM, the samples were critical-point dried (Balzers, Germany) and gold-sputter coated (Sputter Coated S150B, Edwards, United Kingdom’s).

Statistics software NCSS97 was applied to evaluate statistical significance of the data (p < 0.05, ANOVA).

Sponceram HA exhibits a disordered porous structure of coarse grains (compare Fig. 3 (1)). The mechanical durability of the material is low, even a conventional test of the surface roughness in contact mode resulted in low damage. Although the processing of this commercial material is not known in detail, it can be assumed that the calcination process of the porcine bone formed large agglomerates which contain most likely some organic residue. Therefore, sintering of the Sponceram HA powder compacts leads to weakly bonded grains with inter and intraporosity. The surface of the received Sponceram HA exhibits furthermore a wavy structure with large local defects – presumable, this surface reveals weak points of the manufacturing process.

In a preliminary study, conventional grinding/polishing or ultrasonic milling using abrasive B4C particles, respectively, failed to create smooth and homogeneous surfaces [25]. Due to the local abrasive forces, fracturing along the large agglomerates occurred here once again resulting in crushing of the whole samples. Even soft materials like plain paper and polymer sheets as grinding tools did not reveal defect free surfaces. Still, these techniques could be used in order to modify the surface topography in the direction of smooth (using plain paper) or structured (using rough polymer sheets) compared to the rough and inhomogeneous untreated carrier as shown in Fig. (2).

In previous studies, we observed a poor adhesive strength between carrier and chondrocytes recovered from alginate gel (step d). It was found that cells grown on the carrier as a first monolayer are necessary to improve the bonding before re-differentiated chondrocytes are added [31]. Hence, the formation of a cell layer on top of the modified carrier surfaces (step b) was investigated by scanning electron microscopy after two weeks of cultivation.

In Fig. (3) differences in the development of the cell layer could be observed, dependent on the surface structure of the hydroxylapatite carrier. While the HA carrier as received shows a closed cell layer, only a low cell number is visible on the top of the carrier grinded with plain paper or ink jet polymer sheet foil.

After evaluation of the developed cell layer, cartilage-carrier-constructs have been prepared using carriers with different surface structures. In addition, tissue engineered cartilage was generated without using any carrier.

The largest constructs were found for the cultivation without using a carrier which is shown by significantly higher wet weights and heights (Fig. 4). These constructs exhibited also the significantly highest Young’s Modulus of 0.052 MPa. Constructs using carriers grinded with plain paper or with ink jet foil showed higher biomechanical properties than those using untreated carriers. However, only the height of the cartilage-carrier-constructs grinded with paper yielded in a significant difference to the untreated carriers.

The significantly highest glycosaminoglycan content and GAG to DNA ratio have been reached for cartilage-constructs using no carrier (Fig. 5). The DNA concentration is significantly lower compared to the cultivation with carriers. Differences in the biochemical parameters could be hardly observed when comparing cartilage-carrier-constructs with modified surface topographies among each other.

Even though the histological sections in Fig. (6) showed a tissue without any wholes in all cases, only constructs cultured without any carriers developed homogenous tissue in which chondrocytes were evenly distributed. Most notably, in constructs grown on the top of an untreated carrier glycosaminoglycans are unequally distributed.

The immunohistological images (Fig. 7) show a more prominent staining for collagen type II than for collagen type I for all culture conditions. Again, the staining of the unsupported cartilage construct is uniform, while areas with more or less collagen type I and II appeared in constructs cultured with carriers.

In addition, the adhesive strength between carrier and tissue was estimated by applying subjective values. Cartilage was detached from the biomaterial with the help of tweezers and different adhesions were noted. The connection between tissue and biomaterial was weaker for constructs on top of carriers grinded with paper and ink jet foil than for untreated carriers.