Seeding of mesenchymal stem cells into inner part of interconnected porous biodegradable scaffold by a new method with a filter paper.

An appropriate physical support provided by scaffolds creates a supportive environment that directs proliferation and differentiation of stem cells. However, it is difficult to homogenously inoculate stem cells into the inner part of scaffolds at high cell densities. In this study, mesenchymal stem cells were seeded into a hydroxyapatite/poly (D, L-lactic-co-glycolic acid) (HAP/PLGA) scaffold that had enough mechanical strength and porous 3-D structure. With an aid of a filter paper placed under the bottom of a HAP/PLGA block, the cells suspended in a culture medium flowed from the top to the bottom through interconnected pores in the scaffold, and distributed almost homogenously, as compared to cell distribution near the surface of the block by the conventional method using centrifugation or reduced pressure. This simple method with a filter paper may be useful in preparation of cell-scaffold complexes for tissue engineering.

Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold.

Mesenchymal stem cells (MSCs) are multipotent cells capable of proliferating and differentiating into several lineages. In regenerative medicine, their potential as a resource for tissue-replacement therapy is receiving much attention. However, transplanting MSCs to repair larger bone defects in animal models has so far proved disappointing. Here we report on the healing of both critical-sized (5 mm) and massive (15 mm) full-thickness femur defects in rats by implanting a uniquely fabricated PLGA scaffold seeded with MSCs pre-differentiated in vitro into cartilage-forming chondrocytes (MSC-DCs). This strategy closely mimics endochondral ossification, the process by which long bones develop in nature. It is thought that because the transplanted MSC-DCs induced natural bone formation, the defect size was not critical to the outcome. Crucially, after 8 weeks the mean biomechanical strength of femora with the massive 15 mm implant reached 75% that of a normal rat femur, while in the case of 5 mm implants there was no significant difference. Successful healing was also highly reproducible, with bone union occurring in all treated animals examined radiologically 8 or 16 weeks after surgery.

Establishment of reproducible, critical-sized, femoral segmental bone defects in rats.

Development of new treatment methodologies for bone nonunion requires validated experimental models for their application into tissue engineering approaches. Critical-sized bone defect (CSBD) models identify the smallest size at which tissue defects fail to completely heal during the experimental period. The purpose of this study was to systematically determine a CSBD in rat femurs using external fixation. Thirty Fischer 344 rats were equally divided into six groups. Bone defects of 1, 2, 3, 4, 5, and 6 mm were created in the diaphysis of the femur, before external fixation. Defects were evaluated radiographically at weekly intervals and histologically at the predefined healing period of 8 weeks. Defects of 4 mm or more failed to achieve union in our model. Nonunions were characterized by the absence of a bridging callus and filling of the defect with fibrous and muscle tissue. Radiographically, bone ends in nonunions were rounded up to 4 weeks postsurgery. Our systematic approach for identification of a CSBD in rat femurs, revealed that externally fixed defects of 4 mm and larger, failed to heal within the 8-week time frame.

Evaluation of trabecular bone formation in a canine model surrounding a dental implant fixture immobilized with an antimicrobial peptide derived from histatin.

JH8194 induces osteoblast differentiation, although it was originally designed to improve antifungal activity. This suggests that JH8194 is useful for implant treatment. Therefore, the aim of this study was to evaluate the osseointegration capacity of JH8194-modified titanium dental implant fixtures (JH8194-Fi). The implants were randomly implanted into the edentulous ridge of dog mandibles. Healing abutments were inserted immediately after implant placement. Three weeks later, peri-implant bone levels, the first bone-to-implant contact points, and trabecular bone formation surrounding the implants were assessed by histological and digital image analyses based on microcomputed tomography (microCT). The histological analysis revealed an enhancement of mature trabecular bone around the JH8194-Fi compared with untreated fixtures (control-Fi). Similarly, microCT combined with analysis by Zed View™ also showed increased trabecular bone formation surrounding the JH8194-Fi compared with the control-Fi (Student's t-test, P < 0.05). JH8194 may offer an alternative biological modification of titanium surfaces to enhance trabecular bone formation around dental implants, which may contribute to the transient acquirement of osseointegration and the long-term success of implant therapy.

The effect of synthetic octacalcium phosphate in a collagen scaffold on the osteogenicity of mesenchymal stem cells.

Although the efficacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-week-old male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix efficiently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition.

Mesenchymal stem cell-induced cranial suture-like gap in rats.

BACKGROUND: Mesenchymal stem cells are used in the reconstruction of many organs and tissues. However, there are no data regarding cranial suture regeneration using mesenchymal stem cells. Because cranial sutures are important for cranial bone growth, the authors examined whether mesenchymal stem cells could aid with suture reformation in adolescent rats. METHODS: Mesenchymal stem cells were isolated from bone marrow of rat femora. Twenty 4-week-old Fischer 344 male rats received sagittal suture and bone defects with a diameter of 6.0 mm, and mesenchymal stem cells were grafted with membranes. Twenty control rats underwent empty membrane transplantation. The mesenchymal stem cell and control groups were killed at 4, 8, 16, and 24 weeks after surgery. The parietal bones, including the sagittal suture, were observed under a light microscope. Bone structures were measured on digitized photomicrographs captured in a computer. For each sample, bone and suture regeneration were observed by dorsoventral cephalograms. RESULTS: In 4- and 8-week control and mesenchymal stem cell groups, a small volume of new bone formation was observed. In the 16- and 24-week mesenchymal stem cell groups, a large amount of new bone formation and a suture-like gap were identified. In contrast, only a small volume of new bone formation along with craniosynostosis was detected in the 24-week control group. CONCLUSION: These results suggest that mesenchymal stem cell grafting may be a novel option for cranioplasty, not only repairing bone tissue but also resulting in suture-like gap formation, which may advance cranial bone growth.

Selection of common markers for bone marrow stromal cells from various bones using real-time RT-PCR: effects of passage number and donor age.

Bone marrow stromal cells (BMSCs) are valuable in tissue engineering and cell therapy, but the quality of the cells is critical for the efficacy of therapy. To test the quality and identity of transplantable cells, we identified the molecular markers that were expressed at higher levels in BMSCs than in fibroblasts. Using numerous BMSC lines from tibia, femur, ilium, and jaw, together with skin and gum fibroblasts, we compared the gene expression profiles of these cells using DNA microarrays and low-density array cards. The differentiation potential of tibia and femur BMSCs was similar to that of iliac BMSCs, and different from jaw BMSCs, but all BMSC lines had many common markers that were expressed at much higher levels in BMSCs than in fibroblasts; several BMSC markers showed discrete expression patterns between jaw and other BMSCs. The common markers are probably useful in routine tests, but their efficacy may depend upon the passage number or donor age. In our study the passage number markedly altered the expression levels of several markers, while donor age had little effect on them. Considering the effects of in vivo location of BMSCs and passage, magnitude of increase in expression levels, and interindividual differences, we identified several reliable markers -- LIF, IGF1, PRG1, MGP, BMP4, CTGF, KCTD12, IGFBP7, TRIB2, and DYNC1I1 -- among many candidates. This marker set may be useful in a routine test for BMSCs in tissue engineering and cell therapy.