Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model.

OBJECTIVE: To investigate the ability of cell-laden bilayered hydrogels encapsulating chondrogenically and osteogenically (OS) pre-differentiated mesenchymal stem cells (MSCs) to effect osteochondral defect repair in a rabbit model. By varying the period of chondrogenic pre-differentiation from 7 (CG7) to 14 days (CG14), the effect of chondrogenic differentiation stage on osteochondral tissue repair was also investigated. METHODS: Rabbit MSCs were subjected to either chondrogenic or osteogenic pre-differentiation, encapsulated within respective chondral/subchondral layers of a bilayered hydrogel construct, and then implanted into femoral condyle osteochondral defects. Rabbits were randomized into one of four groups (MSC/MSC, MSC/OS, CG7/OS, and CG14/OS; chondral/subchondral) and received two similar constructs bilaterally. Defects were evaluated after 12 weeks. RESULTS: All groups exhibited similar overall neo-tissue filling. The delivery of OS cells when compared to undifferentiated MSCs in the subchondral construct layer resulted in improvements in neo-cartilage thickness and regularity. However, the addition of CG cells in the chondral layer, with OS cells in the subchondral layer, did not augment tissue repair as influenced by the latter when compared to the control. Instead, CG7/OS implants resulted in more irregular neo-tissue surfaces when compared to MSC/OS implants. Notably, the delivery of CG7 cells, when compared to CG14 cells, with OS cells stimulated morphologically superior cartilage repair. However, neither osteogenic nor chondrogenic pre-differentiation affected detectable changes in subchondral tissue repair. CONCLUSIONS: Cartilage regeneration in osteochondral defects can be enhanced by MSCs that are chondrogenically and osteogenically pre-differentiated prior to implantation. Longer chondrogenic pre-differentiation periods, however, lead to diminished cartilage repair.

Osteoblast response to polymethyl methacrylate bioactive glass composite.

Polymethylmethacrylate (PMMA) has been used in many orthopedic and dental applications since the 1960s. Biocompatibility of newly developed surface porous fiber reinforced (SPFR) PMMA based composite has not been previously proven in cell culture environment. Analysis of rat bone marrow stromal cells grown on the different test materials showed only little difference in normalized cell activity or bone sialoprotein (BSP) production between the test materials, but the osteocalcin (OC) levels remained higher (P < 0.015-0.005) through out the test with SPFR-material when compared to tissue culture poly styrene (TCPS). The cells grown on SP-FRC material also showed highest calcium depletion from the culture medium (P < 0.026-0.001) when compared to all other test substrates. SEM images of the cultured samples confirmed that all the materials enabled cell spreading and growth on their surface, but the roughened surface remarkably enhanced this process of cell attachment, division and calcified nodule formation. This study shows that the SP-FRC composite material does not elicit harmful/toxic reactions in cell cultures more than neutral TCPS and can be considered biocompatible. The material possesses good capabilities to form new mineralized tissue onto its surface, and through that a possibility to bond directly to bone. Rough surface seems to enhance osteoblast proliferation and formation of mineralized extracellular matrix.

Crosslinked poly(epsilon-caprolactone/D,L-lactide)/bioactive glass composite scaffolds for bone tissue engineering.

A series of elastic polymer and composite scaffolds for bone tissue engineering applications were designed. Two crosslinked copolymer matrices with 90/10 and 30/70 mol % of epsilon-caprolactone (CL) and D,L-lactide (DLLA) were prepared with porosities from 45 to 85 vol % and their mechanical and degradation properties were tested. Corresponding composite scaffolds with 20-50 wt % of particulate bioactive glass (BAG) were also characterized. Compressive modulus of polymer scaffolds ranged from 190+/-10 to 900+/-90 kPa. Lactide rich scaffolds absorbed up to 290 wt % of water in 4 weeks and mainly lost their mechanical properties. Caprolactone rich scaffolds absorbed no more than 110 wt % of water in 12 weeks and kept their mechanical integrity. Polymer and composite scaffolds prepared with P(CL/DLLA 90/10) matrix and 60 vol % porosity were further analyzed in simulated body fluid and in osteoblast culture. Cell growth was compromised inside the 2 mm thick three-dimensional scaffold specimens as a static culture model was used. However, composite scaffolds with BAG showed increased osteoblast adhesion and mineralization when compared to neat polymer scaffolds.

Biodegradable composite scaffolds incorporating an intramedullary rod and delivering bone morphogenetic protein-2 for stabilization and bone regeneration in segmental long bone defects.

In this study, a two-part bone tissue engineering scaffold was investigated. The scaffold consists of a solid poly(propylene fumarate) (PPF) intramedullary rod for mechanical support surrounded by a porous PPF sleeve for osseointegration and delivery of poly(dl-lactic-co-glycolic acid) (PLGA) microspheres with adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). Scaffolds were implanted into critical size rat segmental femoral defects with internal fixation for 12 weeks. Bone formation was assessed throughout the study via radiography, and following euthanasia, via microcomputed tomography and histology. Mechanical stabilization was evaluated further via torsional testing. Experimental implant groups included the PPF rod alone and the rod with a porous PPF sleeve containing PLGA microspheres with 0, 2 or 8 µg of rhBMP-2 adsorbed onto their surface. Results showed that presence of the scaffold increased mechanical stabilization of the defect, as evidenced by the increased torsional stiffness of the femurs by the presence of a rod compared to the empty defect. Although the presence of a rod decreased bone formation, the presence of a sleeve combined with a low or high dose of rhBMP-2 increased the torsional stiffness to 2.06 ± 0.63 and 1.68 ± 0.56 N·mm, respectively, from 0.56 ± 0.24 N·mm for the rod alone. The results indicate that, while scaffolds may provide structural support to regenerating tissues and increase their mechanical properties, the presence of scaffolds within defects may hinder overall bone formation if they interfere with cellular processes.

Adhesion and proliferation of human fibroblasts on sol-gel coated titania.

The objective of this study was to evaluate growth and attachment of human gingival fibroblasts on nonresorbable sol-gel-derived nanoporous titania (TiO2) coated discs and noncoated commercially pure titania (cpTi) discs in vitro. The strength of attachment was evaluated using serial trypsinization. The number of cells detached from TiO2-substrates was 30% +/- 3%, whereas those detached from the cpTi was 58% +/- 4% indicating a stronger cell attachment on the coated surfaces. In scanning electron microscopy (SEM) images fewer cells, with more rounded shape, were seen with cpTi than with TiO2 after the detachment assay. Fibroblasts grew more efficiently on TiO2 than on cpTi substrates, showing significantly higher cell activities at all times. In transmission electron microscopy (TEM), a continuous layer of two to three cells thick covered the coated and noncoated discs after 7 days of culture. The plasma membrane of cells in contact with the coating was in close opposition and the cytoplasm was ultrastructurally similar to the cells grown on noncoated discs with well-preserved organelles. In conclusion, we demonstrated that the sol-gel-derived TiO2 coatings can facilitate cell growth and attachment of human gingival fibroblasts on titanium in vitro. This in vitro study is in line with our previous in vivo observations of improved soft tissue attachment of TiO2 coatings in comparison with cpTi.