Coral Scaffold Research Articles

De Novo Reconstruction of Functional Bone by Tissue Engineering in the Metatarsal Sheep Model

Large bone defects are still a challenge to orthopedic surgeons. In this study, a massive bone defect with a clinically relevant volume was efficiently reconstructed by transplanting an engineered bone in which mesenchymal stem cells (MSCs) expanded in autologous serum (AS) were combined with a porous scaffold. In the first step, we established that the way in which the MSCs are distributed over the scaffold affects the ultimate bone-forming ability of the transplant: constructs consisting of a natural coral scaffold and a pseudo-periosteal layer of MSCs surrounding the implant (coral-MSC3D) formed significantly more bone than constructs in which the MSCs were distributed throughout the implant (p = 0.01). However, bone healing occurred in only one sheep, owing to the high resorption rate of natural coral scaffold. To overcome this problem, constructs in which MSCs were combined with a porous coralline-based hydroxyapatite (CHA) scaffold having the same architecture as natural coral but a lower resorption rate were prepared. After their implantation, these constructs were found to have the same osteogenic potential as autologous bone grafts in terms of the amount of newly formed bone present at 4 months (p = 0.89) and to have been completely replaced by newly formed, structurally competent bone within 14 months. Nevertheless, although the rate of bone healing was strikingly improved when CHA-MSC3D constructs were used (five of seven animals healed) as compared with the coral-MSC3D construct (one of seven healed), it was still less satisfactory than that obtained with autografts (five of five healed).

Anchoring Dental Implant in Tissue-Engineered Bone Using Composite Scaffold: A Preliminary Study in Nude Mouse Model

The purpose of this study was to fabricate a tissue-engineered bone graft anchoring dental implant with bone marrow stromal cell (bMSC) seeded coral-implant composite scaffold. Titanium dental implants (3 mm in diameter) were inserted into the cylinder coral scaffolds (5 mm in diameter and 1 mm in wall thickness). bMSCs were isolated from iliac bone marrow of adult New Zealand White rabbits, induced by dexamethasone and seeded into the composite scaffold at the density of 2 x 10 8 /mL in 200 muL medium. Nine cell coral-implant complexes were incubated in vitro for 5 days. One complex was processed for scanning electronic microscopy. The other 8 complexes, together with 4 coral scaffold without cell acting as control, were implanted subcutaneously into nude mice back. At 1 and 2 months after implantation, 4 specimens from the experiment group and 2 specimens from the control group were harvested respectively. New bone restoration and new bone integration with dental implant were evaluated by gross inspection, manual handling test, radiographic examination, and histologic observation. Specimens harvested at 2 months after implantation were red and similar to native bone. Manual handling test showed that dental implants were fixed in the newly formed bone. Radiographic examination showed that most of the coral scaffold had been absorbed. Bone density x-ray shadow could be observed around the dental implant. Histologic examination showed that large amount of new bone formed around the dental implants and integrated well with the implants in some area. In the control group no bone formation was observed both macroscopically and microscopically. The results of the study suggested that the tissue-engineered bone of bMSCs seeded natural coral-implant composite scaffold is promising for dental implant anchoring, which has positive implication for clinical jaw reconstruction.

Evolution of an in vivo bioreactor

The ideal bone graft substitute requires osteoconductive, osteoinductive, and osteogenic components. This study introduces an “in vivo bioreactor,” a model in which pluripotent cells are recruited from circulating blood to a vascularized coralline scaffold supplemented with bone morphogenetic protein-2 (BMP-2). The bioreactor generates new, ectopic host bone with the capability of vascularized tissue transfer. More importantly, bone is reproducibly formed in a closed and malleable environment. In a rat model, the superficial inferior epigastric vessels were isolated, ligated, and then threaded through a prefabricated coral cylinder (hydroxyapatite, ProOsteon 500). Experimental groups were characterized by the following variables: (1) with/without incorporation of vascular pedicle; (2) with/without addition of BMP-2 (0.02 mg/cm 3). Scaffolds were harvested 6 weeks after implantation, embedded and sectioned. Tissue samples were decalcified, fixed, and stained with H&E, trichrome green, and CD31/PECAM-1 (a marker of endothelial cells). Vascularized coral scaffolds supplemented with BMP-2 presumably recruited circulating mesenchymal stem cells to generate bone. Bone formation was quantified through histological analysis, and reported as a percentage, area bone/area cross section scaffold × 100. Mean bone formation was 11.30% ± 1.19. All scaffolds supplied by the vascular pedicle, regardless of BMP-2 supplementation, demonstrated neo-vascular ingrowth. Scaffolds lacking a pedicle showed no evidence of vascular ingrowth or bone formation. This paper introduces a model of a novel “in vivo bioreactor” that has future clinical and research applications. The tissue engineering applications of the “bioreactor” include treatment of skeletal defects (nonunion, tumor post-resection reconstruction). The bioreactor also may serve as a unique model in which to study primary and metastatic cancers of bone.

Marrow-derived osteoblasts seeded into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: experimental study in rabbits

To find out if it was possible to prefabricate bone graft in the shape of a human mandibular ramus that possessed a pedicle that carried blood. The pore size of the natural coral was about 200 μm with a porosity of about 36%. The natural coral was made into the shape of a human mandibular ramus. Marrow-derived osteoblasts were seeded into porous natural coral scaffolds in a density of 2 × 108/mL in 300 μL cell suspension. After two days of in vitro incubation, five cell–coral complexes were implanted into cell donor rabbits under the inferior epigastric blood vessels to prefabricate a vascularised bone graft of specific shape. Two months later the bone formation was observed by gross inspection and histological examination. Two months after operation, a well-vascularised bone graft in the shape of the initial coral scaffold and with a blood-carrying pedicle had been regenerated successfully. Osteogenesis followed the pattern of endochondral bone formation. New bone could be seen on the surface and in the pores of coral on histological examination. We have shown that it is possible to fabricate vascularised bone graft in a predetermined shape using tissue engineering. This kind of bone graft may have future clinical application. To find out if it was possible to prefabricate bone graft in the shape of a human mandibular ramus that possessed a pedicle that carried blood. The pore size of the natural coral was about 200 μm with a porosity of about 36%. The natural coral was made into the shape of a human mandibular ramus. Marrow-derived osteoblasts were seeded into porous natural coral scaffolds in a density of 2 × 108/mL in 300 μL cell suspension. After two days of in vitro incubation, five cell–coral complexes were implanted into cell donor rabbits under the inferior epigastric blood vessels to prefabricate a vascularised bone graft of specific shape. Two months later the bone formation was observed by gross inspection and histological examination. Two months after operation, a well-vascularised bone graft in the shape of the initial coral scaffold and with a blood-carrying pedicle had been regenerated successfully. Osteogenesis followed the pattern of endochondral bone formation. New bone could be seen on the surface and in the pores of coral on histological examination. We have shown that it is possible to fabricate vascularised bone graft in a predetermined shape using tissue engineering. This kind of bone graft may have future clinical application.

Marrow-derived osteoblasts seeded into porous natural coral to prefabricate a vascularised bone graft in the shape of a human mandibular ramus: experimental study in rabbits

To find out if it was possible to prefabricate bone graft in the shape of a human mandibular ramus that possessed a pedicle that carried blood. The pore size of the natural coral was about 200 microm with a porosity of about 36%. The natural coral was made into the shape of a human mandibular ramus. Marrow-derived osteoblasts were seeded into porous natural coral scaffolds in a density of 2 x 10(8)/mL in 300 microL cell suspension. After two days of in vitro incubation, five cell-coral complexes were implanted into cell donor rabbits under the inferior epigastric blood vessels to prefabricate a vascularised bone graft of specific shape. Two months later the bone formation was observed by gross inspection and histological examination. Two months after operation, a well-vascularised bone graft in the shape of the initial coral scaffold and with a blood-carrying pedicle had been regenerated successfully. Osteogenesis followed the pattern of endochondral bone formation. New bone could be seen on the surface and in the pores of coral on histological examination. We have shown that it is possible to fabricate vascularised bone graft in a predetermined shape using tissue engineering. This kind of bone graft may have future clinical application.

Bone graft in the shape of human mandibular condyle reconstruction via seeding marrow-derived osteoblasts into porous coral in a nude mice model

Purpose: The purpose of this study was to develop a tissue-engineered bone graft model in the shape of a human mandibular condyle. Materials and Methods: Natural coral with a pore size of 150 to 220 μm and porosity of about 36% was molded into the shape of a human mandibular condyle. Culture-expanded rabbit marrow mesenchymal stem cells were induced by recombinant human bone morphogenetic protein-2 (rhBMP2) to improve osteoblastic phenotype. Then marrow-derived osteoblasts were seeded into natural coral at a density of 2 × 108/mL and incubated in vitro for 3 days before implantation. The cell–coral complexes were implanted subcutaneously into the backs of nude mice and incubated in vivo for 2 months before harvesting. Implantation of coral alone acted as control. The specimens were processed for gross inspection, radiographic examination, and histologic and scanning electronic microscopic observation. Results: The results showed that new bone grafts in the shape of a human mandibular condyle were successfully developed 2 months after implantation and maintained the initial shape of the natural coral scaffold. New bone could be observed histologically on the surface and in the pores of natural coral in all specimens in the cell-seeding group (6 of 6), whereas in the control group there was no evidence of osteogenesis process (0 of 4). Conclusion: This study suggests the feasibility of using porous coral as scaffold material transplanted with marrow-derived osteoblasts to restore bone graft in the shape of human mandibular condyle and shows the potential of using this method for the reconstruction of bone defects. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:1155-1159, 2002

In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye.

Recent advances in the field of tissue engineering have culminated in new tissue substitutes that combine a biomaterial and precursor cells. The effectiveness of these materials is generally assessed in animals, but few studies explore the fate of the transplanted cells in vivo, despite its paramount importance for understanding the function of the engineered tissues. Current methods that use reporter genes or chimeric animals are not always well suited to solving tissue-engineering problems. We therefore developed a new method for irreversible labeling of cells to track their fate in vivo. We used a fluorescent lipophilic probe, CM-Dil, that avidly binds to the cell membrane. Human bone marrow stromal fibroblasts could be labeled with 20 microM CM-Dil in 30 min. The CM-Dil was not cytotoxic and did not affect cell proliferation in vitro. Cells could be monitored for up to 30 days when placed in a coral scaffold and implanted intramuscularly or in a bony site. However, the fluorescence intensity decreased roughly in parallel with the number of cell divisions. This fact needs to be taken into account during the design and interpretation of experiments. We believe that this technique is also of interest for other cell types.

Tissue-engineered bone regeneration

Bone lesions above a critical size become scarred rather than regenerated, leading to nonunion. We have attempted to obtain a greater degree of regeneration by using a resorbable scaffold with regeneration-competent cells to recreate an embryonic environment in injured adult tissues, and thus improve clinical outcome. We have used a combination of a coral scaffold with in vitro-expanded marrow stromal cells (MSC) to increase osteogenesis more than that obtained with the scaffold alone or the scaffold plus fresh bone marrow. The efficiency of the various combinations was assessed in a large segmental defect model in sheep. The tissue-engineered artificial bone underwent morphogenesis leading to complete recorticalization and the formation of a medullary canal with mature lamellar cortical bone in the most favorable cases. Clinical union never occurred when the defects were left empty or filled with the scaffold alone. In contrast, clinical union was obtained in three out of seven operated limbs when the defects were filled with the tissue-engineered bone.