Repair of large osteochondral defects in rabbits using porous hydroxyapatite/collagen (HAp/Col) and fibroblast growth factor-2 (FGF-2).

Articular cartilage has a limited capacity for self-renewal. This article reports the development of a porous hydroxyapatite/collagen (HAp/Col) scaffold as a bone void filler and a vehicle for drug administration. The scaffold consists of HAp nanocrystals and type I atelocollagen. The purpose of this study was to investigate the efficacy of porous HAp/Col impregnated with FGF-2 to repair large osteochondral defects in a rabbit model. Ninety-six cylindrical osteochondral defects 5 mm in diameter and 5 mm in depth were created in the femoral trochlear groove of the right knee. Animals were assigned to one of four treatment groups: porous HAp/Col impregnated with 50 microl of FGF-2 at a concentration of 10 or 100 microg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 microl of PBS (HAp/Col group); and no implantation (defect group). The defect areas were examined grossly and histologically. Subchondral bone regeneration was quantified 3, 6, 12, and 24 weeks after surgery. Abundant bone formation was observed in the HAp/Col implanted groups as compared to the defect group. The FGF10 group displayed not only the most abundant bone regeneration but also the most satisfactory cartilage regeneration, with cartilage presenting a hyaline-like appearance. These findings suggest that porous HAp/Col with FGF-2 augments the cartilage repair process.

Effects of gamma-ray irradiation on mechanical properties, osteoconductivity, and absorption of porous hydroxyapatite/collagen.

In this study, the effects of gamma-ray irradiation on the mechanical properties, absorbability, and osteoconductivity of porous hydroxyapatite/collagen (HAp/Col) were investigated. Porous HAp/Col was exposed to 16, 25, 35, or 50 kGy of gamma-ray irradiation. The compressive elastic modulus showed irradiation dose-dependence, with a particularly pronounced decrease in the 50-kGy treatment group. An in vitro enzymatic digestion test showed that gamma-ray irradiation of porous HAp/Col resulted in accelerated degradation by collagenase. For in vivo studies, porous HAp/Col was transplanted into the back muscles or bone defects in the femoral condyle of rats. Specimens were obtained at 2, 4, and 8 weeks postoperatively. Absorption of the implants in the muscle was time- and irradiation dose-dependent, with notable absorption for the 35- and 50-kGy groups at 2 weeks. At the skeletal sites, porous HAp/Col demonstrated high osteoconductivity in all irradiation treatment groups. Interestingly, not only implant absorption but also bone formation was irradiation dose-dependent at early time points.

Isolation of osteogenic progenitor cells from trabecular bone for bone tissue engineering.

Trabecular bone fragments can be percutaneously harvested from the ilium using methods that are similar in invasiveness to aspiration of bone marrow. In this study, we investigated the use of the trabecular bone as a cell source for bone tissue engineering. Trabecular bone-derived progenitor cells (TB cells) were isolated with a simple method in which trabecular fragments were first cultured as explants, and then the cells were released by trypsin digestion and advanced to a monolayer culture. The properties of TB cells prepared in this procedure were compared with bone marrow-derived progenitor cells (BM cells). A large number of TB cells could be obtained with less variation among donors, compared with BM cells. In multiple harvests of donor tissue through the same aspiration hole at the cortex, TB cells could be more consistently obtained in primary culture. The proliferative potential of BM and TB cells was similar in serial subculture. TB cells showed a higher alkaline phosphatase expression in the surface marker analysis and greater in vitro osteogenic abilities than BM cells after the initial 14 days of culture. In in vivo bone formation studies, TB cells also showed a higher osteogenic potential than BM cells. The results of this study suggest that TB cells can be considered an attractive source for clinical bone regeneration.

Bone regeneration with autologous plasma, bone marrow stromal cells, and porous beta-tricalcium phosphate in nonhuman primates.

To potentiate the bone formation capability of bone marrow stromal cell (BMSC)/beta-tricalcium phosphate (beta-TCP) constructs, we devised an autologous plasma-based construct. We tested its effectiveness and investigated the effects of its components on a monkey ectopic bone formation model. The autologous plasma (platelet-rich plasma, PRP, or platelet-poor plasma, PPP)/BMSC/beta-TCP construct (R group or P group) showed significantly more bone formation at 3 and 6 weeks after implantation than a conventional BMSC/beta-TCP construct using a culture medium (M group). There was no significant difference between the P and R groups. Moreover, the P group constructs with a 10-fold lower cell concentration yielded equivalent bone formation to the M group at 5 weeks after implantation. To elucidate the effect of fibrin and serum contained in the plasma, five constructs were prepared using the following cell vehicles: autologous serum + fibrinogen (0, 1, 4, or 16 mg/mL) or phosphate-buffered saline + fibrinogen (4 mg/mL). The serum + fibrinogen (4 mg/mL, physiological concentration of monkeys) construct showed the most abundant bone formation at 3 weeks after implantation, though at 5 weeks no statistical difference existed among the groups. Autologous plasma efficiently promoted osteogenesis of BMSCs/porous beta-TCP constructs, and both fibrin and serum proved to play significant roles in the mechanism.

Effects of continuous dexamethasone treatment on differentiation capabilities of bone marrow-derived mesenchymal cells.

Human bone marrow-derived mesenchymal cells (hBMMCs) originate from cell populations in the bone marrow and are capable of differentiating along multiple mesenchymal lineages. To differentiate hBMMCs into osteoblasts, adipocytes and chondrocytes, dexamethasone has been used as a differentiation reagent. We hypothesized that dexamethasone would augment the responsiveness of BMMCs to other differentiation reagents and not define the lineage. This study investigated the effect of continuous treatment with 100 nM dexamethasone on the differentiation of BMMCs into three different lineages. hBMMCs cultured with continuous dexamethasone treatment (100 nM) exhibited higher mRNA expression levels of osteogenic markers and higher positive rates of colony forming unit assays for osteogenesis compared to hBMMCs treated with dexamethasone only during the differentiation culture. Furthermore, continuous dexamethasone treatment augmented bone formation capability of monkey-derived BMMCs in a bone induction experimental model at an extra skeletal site. In addition, continuously dexamethasone-treated hBMMCs formed larger chondrogenic pellets and expressed SOX9 at higher level than the control BMMCs. Likewise, continuous dexamethasone treatment facilitated adipogenic differentiation based on mRNA level and colony forming unit analysis. To investigate the mechanism of the augmentation of differentiation, further studies on apoptosis were conducted. The studies indicated that dexamethasone selectively induced apoptosis of some populations of hBMMCs which were thought to have poor differentiation capability.