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Cells with the desired phenotype and number are critical for regenerative medicine and tissue engineering. Uniparental parthenogenetic embryonic stem cells (pESCs) share fundamental properties with embryonic stem cells. This study aims to determine the viability of pESC-based tissue engineering for bone and cartilage reconstruction. The mouse pESCs were cultured in suspension to form embryoid bodies. An adherent cultivation approach was employed to obtain parthenogenetic embryonic mesenchymal stem cells (pMSCs) from the embryoid bodies. Then, the pMSCs were cultured in conditional media to differentiate into osteogenic and chondrogenic lineages. The pESC-derived osteoblasts and chondroblasts were seeded into coral and sodium alginate scaffolds, respectively. The cell-seeded scaffolds were implanted into dorsal subcutaneous pockets of nude mice to evaluate ectopic reconstruction of bone and cartilage. We demonstrated that pESCs display the capacity to differentiate into all three germ layers. The generated pMSCs were able to differentiate into osteogenic and chondrogenic lineages, which survived well after seeding into coral and alginate acid scaffolds. Six weeks after cell-scaffold implantation, gross inspection and histological examination revealed that ectopic bone and cartilage tissues had successfully regenerated in the specimen. According to the findings of this study, pESC derivatives have a high potential for bone and cartilage regeneration.

Tissue-engineered skin equivalent (TESE) is an optimized alternative for the treatment of skin defects. Designing and fabricating biomaterials with desired properties to load cells is critical for the approach. In this study, we aim to develop a novel TESE with recombinant human collagen (rHC) hydrogel and fibroblasts to improve full-thickness skin defect repair. First, the bioactive effect of rHC on fibroblast proliferation, migration and phenotype was assayed. The results showed that rHC had good biocompatibility and could stimulate fibroblasts migration and secrete various growth factors. Then, rHC was cross-linked with transglutaminase (TG) to prepare rHC hydrogel. Rheometer tests indicated that 10% rHC/TG hydrogel could reach a oscillate stress of 251 Pa and remained stable. Fibroblasts were seeded into rHC/TG hydrogel to prepare TESE. Confocal microscope and scanning electronic microscope observation showed that seeded fibroblasts survived well in the hydrogel. Finally, the therapeutic effect of the newly prepared TESE was tested in a mouse full-thickness skin defect model. The results demonstrated that TESE could significantly improve skin defect repair in vivo. Conclusively, TESE prepared from rHC and fibroblasts in this study exhibits great potential for clinical application in the future.

Background Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. Methods The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Results Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. Conclusions pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue-engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self-renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic-specific markers and an increasing COL1A1:3A1 ratio. The pSC-derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic-co-glycolic) acid scaffolds. Finally, engineered tendon-like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte-scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration.

Tissue-engineered skin (TES) holds great promise for wound healing in the clinic. However, optimized preservation methods remain an obstacle for its wide application. In this experimental work, we developed a novel approach to preserve TES in the desiccated state with trehalose. The uptake of trehalose by fibroblasts under various conditions, including the trehalose concentration, incubation temperature and time, was studied. The cell viability was investigated by the MTT assay and CFSE/PI staining after cryodesiccation and rehydration. TES was then prepared and incubated with trehalose, and the wound healing effect was investigated after desiccated preservation. The results showed that the optimized conditions for trehalose uptake by fibroblasts were incubation in 200 mM trehalose at 37 °C for 8 h. Cryodesiccated cells and TES maintained 37.55% and 28.31% viabilities of controls, respectively. Furthermore, cryodesiccated TES exhibited a similar wound healing effect to normal TES. This novel approach enabled the preservation and transportation of TES at ambient temperature with a prolonged shelf time, which provides great advantages for the application of TES.

Electrospinning is an effective means to generate nano- to micro-scale polymer fibers resembling native extracellular matrix for tissue engineering. However, a major problem of electrospun materials is that limited pore size and porosity may prevent adequate cellular infiltration and tissue ingrowth. In this study, we first prepared thin layers of hydroxyapatite nanoparticle (nHA)/poly-hydroxybutyrate (PHB) via electrospinning. We then laminated the nHA/PHB thin layers to obtain a scaffold for cell seeding and bone tissue engineering. The results demonstrated that the laminated scaffold possessed optimized cell-loading capacity. Bone marrow mesenchymal stem cells (MSCs) exhibited better adherence, proliferation and osteogenic phenotypes on nHA/PHB scaffolds than on PHB scaffolds. Thereafter, we seeded MSCs onto nHA/PHB scaffolds to fabricate bone grafts. Histological observation showed osteoid tissue formation throughout the scaffold, with most of the scaffold absorbed in the specimens 2months after implantation, and blood vessels ingrowth into the graft could be observed in the graft. We concluded that electrospun and laminated nanoscaled biocomposite scaffolds hold great therapeutic potential for bone regeneration.

Tissue-engineered bone provides a promising method for the rehabilitation of acquired bone defects and congenital deformities. However, generating a vascular supply to the engineered graft remains a major challenge. We report a novel strategy to engineer vascularized bone grafts with osteogenic and angiogenic lineage differentiated marrow mesenchymal stem cells (MSCs). MSCs were expanded to form an osteogenic cell sheet using a continuous culture method and a scraping technique under osteogenic culture conditions. Another portion of MSCs was directed to differentiate into highly proliferative endothelial progenitor cells (EPCs), which were then seeded onto the cell sheets. Cell sheet-EPC complexes were implanted subcutaneously in nude mice. Cell sheets without EPCs were also implanted as a control. The mice were sacrificed, and the samples were harvested for evaluation consisting of micro-CT scanning, histological analysis and scanning electronic microscopy 4 and 8 weeks after implantation. The results showed that cell sheets were composed of viable cells and extracellular matrix and showed apparent mineralization. The obtained EPCs could express the specific antigen marker of CD31 and form capillary-like structures in vitro. The osteogenic cell sheet-EPC complexes yielded well-vascularized bone grafts 4 and 8 weeks after implantation. Both bone density and vascular density were significantly higher in the cell sheet-EPC complex group than in the control group. The results demonstrated that the introduction of EPCs could not only generate a vascular network but also increase bone formation for cell sheet-based bone engineering. These findings suggest that the strategy of engineering bone grafts with osteogenic and angiogenic lineage differentiated MSCs has great potential for clinical applications to repair large bone defects.

Background Marrow mesenchymal stem cells (MSCs) can differentiate into specific phenotypes, including chondrocytes, and have been widely used for cartilage tissue engineering. However, cartilage grafts from MSCs exhibit phenotypic alternations after implantation, including matrix calcification and vascular ingrowth. Methods We compared chondromodulin-1 (Chm-1) expression between chondrocytes and MSCs. We found that chondrocytes expressed a high level of Chm-1. We then adenovirally transduced MSCs with Chm-1 and applied modified cells to engineer cartilage in vivo. Results A gross inspection and histological observation indicated that the chondrogenic phenotype of the tissue-engineered cartilage graft was well maintained, and the stable expression of Chm-1 was detected by immunohistological staining in the cartilage graft derived from the Chm-1 gene-modified MSCs. Conclusions Our findings defined an essential role for Chm-1 in maintaining chondrogenic phenotype and demonstrated that Chm-1 gene-modified MSCs may be used in cartilage tissue engineering.

Tissue-engineered blood vessels have mainly relied on endothelial cells (ECs), smooth muscle cells (SMCs), and biocompatible materials. However, long-term results have revealed several material-related failures, such as stenosis, thromboembolization, and the risk of infection. Furthermore, SMCs from elderly persons have reduced capacity in proliferation and collagen production. Mesenchymal stem cells (MSCs) have the ability to differentiate into multiple cell lineages, including osteoblasts, chondrocytes, ECs, and SMCs. In the current experiment, rabbit MSCs were cultured to form a cell sheet. A tissue-engineered vascular graft (TEVG) was fabricated by rolling the MSC sheet around a mandrel. The TEVG was implanted into a defect of the common carotid artery after it was examined macroscopically and microscopically. Hematoxylin and eosin staining showed that cell sheet was composed of five to seven layers of cells with the thickness of 40-50 µm. Results from the adhesion assay revealed that MSCs had similar antiplatelet adhesion property to ECs. Histological analysis of TEVGs showed that the layers of the cell sheet had fully fused in vitro. After implantation, TEVGs had excellent patency and integrated well with the native vessel. The structure of the TEVGs was similar to that of the native artery 4 weeks after implantation. Electron microscopy showed that the implanted TEVGs endothelialized. These results indicated that a completely biological TEVG could be assembled with autologous MSCs. These TEVGs are useful for revascularization in humans, which would reduce the occurrence of complications caused by foreign materials.

With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.

Objectives: The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP). Material and methods: We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous β-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model. Results: The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site. Conclusion: The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings. To cite this article: Ma D, Yao H, Tian W, Chen F, Liu Y, Mao T, Ren L. Enhancing bone formation by transplantation of a scaffold-free tissue-engineered periosteum in a rabbit model. Clin. Oral Impl. Res. 22, 2011; 1193–1199. doi: 10.1111/j.1600-0501.2010.02091.x

Limited donor sites of cartilage and dedifferentiation of chondrocytes during expansion, low tissue reconstruction efficiency, and uncontrollable immune reactions to foreign materials are the main obstacles to overcome before cartilage tissue engineering can be widely used in the clinic. In the current study, we developed a novel strategy to fabricate tissue-engineered trachea cartilage grafts using marrow mesenchymal stem cell (MSC) macroaggregates and hydrolyzable scaffold of polylactic acid-polyglycolic acid copolymer (PLGA). Rabbit MSCs were continuously cultured to prepare macroaggregates in sheet form. The macroaggregates were studied for their potential for chondrogenesis. The macroaggregates were wrapped against the PLGA scaffold to make a tubular composite. The composites were incubated in spinner flasks for 4 weeks to fabricate trachea cartilage grafts. Histological observation and polymerase chain reaction array showed that MSC macroaggregates could obtain the optimal chondrogenic capacity under the induction of transforming growth factor-beta. Engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans. PLGA scaffold degraded totally during in vitro incubation and the engineered cartilage graft was composed of autologous tissue. Based on this novel, MSC macroaggregate and hydrolyzable scaffold composite strategy, ready-to-implant autologous trachea cartilage grafts could be successfully fabricated. The strategy also had the advantages of high efficiency in cell seeding and tissue regeneration, and could possibly be used in future in vivo experiments.

This study investigated the hypothesis that cell sheets composed of multilayered rabbit bone marrow derived mesenchymal stem cells (MSC) could be assembled with two kinds of implants (surface-modified titanium and zirconia) for the construction of a MSC-implant. The MSC sheets were harvested from culture flasks, wrapped around implants to construct the complexes, and then cultured in osteogenic medium. The layered cell sheets integrated well with implants and remained viable, with small mineralized nodules visible on the implant surfaces for up to four weeks after culture. Cells on the implants underwent classical in vitro osteogenic differentiation with an associated elevation of alkaline phosphatase activity and bone- and vascular-related protein expression. In vivo, two kinds of cell sheet-implant complexes were transplanted under the skin of SCID mice and cultured for eight weeks. For the MSC sheet titanium implant complex, histological examination revealed that new bone tissue that formed around implants followed a predominantly endochondral pathway, exhibiting histological markers of native bone; for the MSC sheet zirconia implant complex, however, intramembranous ossification appeared to occur on the surface of the zirconia implant, as observed with typical osteocytes embedded in dense matrix and accompanied by both microvessels and marrow cavities. These findings demonstrate that MSC-implants possessing osteogenic and vascularization abilities can be produced using cell sheet engineering techniques in conjunction with routine implant materials, which provide a novel technology to modify the implant surface.

With the increasing occurrence of vascular diseases and poor long-term patency rates of current small diameter vascular grafts, it becomes urgent to pursuit biomaterial as scaffold to mimic blood vessel morphologically and mechanically. In this study, novel human-like collagen (HLC, produced by recombinant E. coli)/chitosan tubular scaffolds were fabricated by cross-linking and freeze-drying process. The scaffolds were characterized by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), and tensile test, respectively. Human venous fibroblasts were expanded and seeded onto the scaffolds in the density of 1 x 10(5) cells/cm(2). After a 15-day culture under static conditions, the cell-polymer constructs were observed using SEM, confocal laser scanning microscopy (CLSM), histological examination, and biochemical assays for cell proliferation and extracellular matrix production (collagen and glycosaminoglycans). Furthermore, the scaffolds were implanted into rabbits' livers to evaluate their biocompatibility. The results indicated that HLC/chitosan tubular scaffolds (1) exhibited interconnected porous structure; (2) achieved the desirable levels of pliability (elastic up to 30% strain) and stress of 300 +/- 16 kPa; (3) were capable of enhancing cell adhesion and proliferation and ECM secretion; (4) showed superior biocompatibility. This study suggested the feasibility of HLC/chitosan composite as a promising candidate scaffold for blood vessel tissue engineering.

In this study, cell sheets comprising multilayered living bone marrow stromal cells and extracellular matrix were assembled with tubular coral scaffolds for long bone regeneration. Cell sheet with visible mineralized nodules was harvested and wrapped against tubular coral scaffolds with 5 mm diameter and 1.5 mm wall thickness. New bone formation was investigated by CT scan and histological observation 8 and 12 weeks after implantation of cell sheet/scaffold. The results showed that cortical bone formed within the constructs for both groups. New bone composed 25.75% of the graft in 8 weeks group, compared to that of 40.01% in 12 weeks group. Histological examination showed that new bone formation was in the manner of endochondral osteogenesis, with woven bone matrix subsequently maturing into fully mineralized compact bone. These findings demonstrated that osteogenic cell sheet could vitalize tubulate coral scaffolds to regenerate bone graft with similar shape and structure to native bone.

Purpose Repair of nasal and auricular malformation remains an obstacle for clinicians because of poor regenerative capacity of cartilage and limitation of donor sites. In the current study, we developed a novel approach to regenerate implantable nasal alar cartilage by using marrow precursor cell (MPC) sheet and biodegradable scaffold of polylactic acid-polyglycolic acid copolymer (PLGA). Materials and Methods Rabbit MPCs were expanded and induced by transforming growth factor-β to improve chondrocyte phenotype. MPC sheets were obtained by continuous culture and used to wrap PLGA scaffold in the shape of the human nasal alar. The constructs were incubated in a spinner flask for 4 weeks, and cartilage formation was investigated by gross inspection and histological examination. The constructs were then implanted subcutaneously into a nude mouse. Specimens were harvested and analyzed 4 weeks after implantation. Results The results showed that cartilaginous tissue formed and PLGA absorbed during in vitro incubation. Histological analysis showed engineered cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans, and kept the initial shape of the nasal alar. Based on this “MPC sheet combining polymer strategy,” implantable nasal alar could be successfully regenerated. Conclusion This strategy has the advantage of high cell transplantation efficiency and great potential for clinical application.

Our group has recently fabricated 3-dimensional scaffolds of unique architecture to mediate favorable cell binding, proliferation, and differentiation. In this study, the osteoconductive and bioactive polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds were assessed for the treatment of critical-sized defects of the mandible, with respect to new bone formation. Platelet-rich plasma (PRP) was combined with some scaffolds to test if bone regeneration could be enhanced.Autologous PRP was prepared from whole blood collected from 8 mongrel dogs. In each dog, 3 defects (18 x 10 x 7 mm) were created at either the left or right mandible and treated with 1 of 2 treatment modalities: 1) grafting with scaffolds alone; or 2) grafting with scaffolds loaded with PRP. The scaffolds were stabilized using 2 dental implants each to prevent rotation. Micro-CT and histologic analysis were carried out on samples after 6 and 9 months.Micro-CT measurements showed that PRP-treated defects had 98.3% and 58.3% more bone volume fraction than defects grafted with scaffolds alone at 6 and 9 months, respectively (P.05). No significant difference was noted between caudal and frontal situated PRP-treated defects, but a significant difference was observed between male and female dogs. Histologic analyses verified the deposition of osteoid and new bone trabeculae throughout the section at 6 months. The defect margins were filled with mature bone trabeculae at 9 months but the middle section of the scaffolds manifested disturbed mineralization. The scaffolds experienced 33% degradation from 6 to 9 months. PRP treatment had negligible effect on the degradation of the scaffolds.The pilot study showed that the treatment of critical-sized defects of the mandible with PCL-TCP scaffolds may be augmented by the addition of PRP.

Despite of progresses in tissue engineering based on cell/scaffold strategy, uneven cell distribution as well as tissue formation in the scaffold, limited cell seeding efficiency and inflammatory reaction triggered by the degradation of scaffold remain problems to be resolved. In this study, we proposed a novel cell-macroaggregate cultivation system, and explored a feasible strategy to construct three-dimensional cartilage tissue with shape of human nasal alar by using cell macroaggregate. Isolated chondrocytes was cultured at high density to form a monolayer chondrocyte sheet as well as expanded for seeding on the sheet to produce mechanically operable cell macroaggregate. Chondrocyte macroaggregates were then fabricated into transplants with shape of nasal alar by using Internal support or External scaffold techniques; results of in vivo chondrogenesis were investigated in immunocompetent animal. Chondrocyte macroaggregates presented long survival time and good viability; constructs fabricated using both techniques can develop into tissues with characteristic structure of native cartilage, glycosaminoglycans as well as type II collagen were highly produced in the ECM of engineered cartilages. By placing hyaluronan ester film as Internal support, the predetermined shape of the chondrocyte macroaggregate can be well maintained. In contrast, due to the poor mechanical stability of grafts fabricated in External scaffold group, obvious deformation occurred in harvested specimens. The experiment proved the usefulness of chondrocyte macroaggregate in cartilage regeneration, and provided a new strategy to engineer cartilage with special shape by using cell macroaggregate/biodegradable support.

Tissue engineered bone has become a bone substitute for the treatment of bone defects in animal research. This study investigated the osteogenesis capacity of coral-MSCs-rhBMP-2 composite with the auto-bone-graft as control. Coral-MSCs-rhBMP-2 composite were fabricated by coral (as main scaffold), rhBMP-2 (as growth factor), and MSCs (cultured from iliac marrow as seed cells). Critical-sized defects (d = 15 mm) were made on forty rabbits crania and treated by different composite scaffolds: iliac autograft (n = 8), coral (n = 8), rhBMP-2/coral (n = 8), and MSCs/rhBMP-2/coral (n = 8). The defects were evaluated by gross observation, radiographic examination, histological examination, and histological fluorescence examinations after 8 and 16 weeks. The results showed that repair of bone defect was the least in coral group, and significant ingrowth of new bone formation and incorporation could be seen with 77.45% +/- 0.52% in radiopacity in MSCs/rhBMP-2/coral group, which was similar to that in iliac autograft group (84.61% +/- 0.56% in radiopacity). New bone formation in MSCs/rhBMP-2/coral group was more than that in rhBMP-2/coral group. And osteogenesis rate in MSCs/rhBMP-2/coral group (10.23 +/- 1.45 microm) was much faster than that in rhBMP-2/coral group (5.85 +/- 2.19 microm) according to histological fluorescence examination. Newly formed bone partly came from induced MSCs in composite scaffold according to bromodeoxyuridine immunohistochemical examination. These data implicated that MSCs could produce synergic effect with coral-rhBMP-2, and the tissue engineered bone of coral-MSCs-rhBMP-2 is comparable to auto-bone-graft for the repair of critical-sized bone defect.

Cell-sheet techniques have been proven effective in various soft tissue engineering applications. In this experiment, we investigated the feasibility of bone tissue engineering using a hybrid of mesenchymal stem cell (MSC) sheets and PLGA meshes. Porcine MSCs were cultured to a thin layer of cell sheets via osteogenic induction. Tube-like long bones were constructed by wrapping the cell sheet on to PLGA meshes resulting in constructs which could be cultured in spinner flasks, prior to implantation in nude rats. Our results showed that the sheets were composed of viable cells and dense matrix with a thickness of about 80-120 microm, mineral deposition was also observed in the sheet. In vitro cultures demonstrated calcified cartilage-like tissue formation and most PLGA meshes were absorbed during the 8-week culture period. In vivo experiments revealed that dense mineralized tissue was formed in subcutaneous sites and the 8-week plants shared similar micro-CT characteristics with native bone. The neo tissue demonstrated histological markers for both bone and cartilage, indicating that the bone formation pathway in constructs was akin to endochondral ossification, with the residues of PLGA having an effect on the neo tissue organization and formation. These results indicate that cell-sheet approaches in combination with custom-shaped scaffolds have potential in producing bone tissue.

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

The development of bone tissue engineering has provided new solutions for bone defects. However, the cell-scaffold-based approaches currently in use have several limitations, including low cell seeding rates and poor bone formation capacity. In the present study, we developed a novel strategy to engineer bone grafts using mesenchymal stem cell sheets and coral particles. Rabbit bone marrow mesenchymal stem cells were continuously cultured to form a cell sheet with osteogenic potential and coral particles were integrated into the sheet. The composite sheet was then wrapped around a cylindrical mandrel to fabricate a tubular construct. The resultant tubular construct was cultured in a spinner-flask bioreactor and subsequently implanted into a subcutaneous pocket in a nude mouse for assessment of its histological characteristics, radiological density and mechanical property. A similar construct assembled from a cell sheet alone acted as a control. In vitro observations demonstrated that the composite construct maintained its tubular shape, and exhibited higher radiological density, compressive strength and greater extracellular matrix deposition than did the control construct. In vivo experiments further revealed that new bone formed ectopically on the composite constructs, so that the 8-week explants of the composite sheets displayed radiological density similar to that of native bone. These results indicate that the strategy of using a combination of a cell sheet and coral particles has great potential for bone tissue engineering and repairing bone defects.

A higher degree of mineralization is found within scaffold groups implanted with cells compared to scaffold alone demonstrating greater bone regenerative potential of cell-scaffold constructs Tissue engineered bone analysed using ESEM and SAXS demonstrates bone formation within the scaffold to be preferentially aligned around the scaffold struts. The mineral particles are not shown to orientate around the osteons within the native bone.

Tissue-engineered bone provides a promising method for the rehabilitation of acquired bone defects and congenital deformities. However, generating a vascular supply to the engineered graft remains a major challenge. We report a novel strategy to engineer vascularized bone grafts with osteogenic and angiogenic lineage differentiated marrow mesenchymal stem cells (MSCs). MSCs were expanded to form an osteogenic cell sheet using a continuous culture method and a scraping technique under osteogenic culture conditions. Another portion of MSCs was directed to differentiate into highly proliferative endothelial progenitor cells (EPCs), which were then seeded onto the cell sheets. Cell sheet-EPC complexes were implanted subcutaneously in nude mice. Cell sheets without EPCs were also implanted as a control. The mice were sacrificed, and the samples were harvested for evaluation consisting of micro-CT scanning, histological analysis and scanning electronic microscopy 4 and 8 weeks after implantation. The results showed that cell sheets were composed of viable cells and extracellular matrix and showed apparent mineralization. The obtained EPCs could express the specific antigen marker of CD31 and form capillary-like structures in vitro. The osteogenic cell sheet-EPC complexes yielded well-vascularized bone grafts 4 and 8 weeks after implantation. Both bone density and vascular density were significantly higher in the cell sheet-EPC complex group than in the control group. The results demonstrated that the introduction of EPCs could not only generate a vascular network but also increase bone formation for cell sheet-based bone engineering. These findings suggest that the strategy of engineering bone grafts with osteogenic and angiogenic lineage differentiated MSCs has great potential for clinical applications to repair large bone defects.

Clinical application of platelet-rich plasma (PRP)-based injectable tissue engineering is limited by weak mechanical properties and a rapid fibrinolytic rate. We proposed a new strategy, a cell bricks-stabilized PRP injectable system, to engineer and regenerate cartilage with stable morphology and structure in vivo. Chondrocytes from the auricular cartilage of rabbits were isolated and cultured to form cell bricks (fragmented cell sheet) or cell expansions. Fifteen nude mice were divided evenly (n = 5) into cells-PRP (C-P), cell bricks-PRP (CB-P) and cell bricks-cells-PRP (CB-C-P) groups. Cells, cell bricks or a cell bricks/cells mixture were suspended in PRP and were injected subcutaneously in animals. After 8 weeks, all the constructs were replaced by white resilient tissue; however, specimens from the CB-P and CB-C-P groups were well maintained in shape, while the C-P group appeared distorted, with a compressed outline. Histologically, all groups presented lacuna-like structures, glycosaminoglycan-enriched matrices and positive immunostaining of collagen type II. Different from the uniform structure presented in CB-C-P samples, CB-P presented interrupted, island-like chondrogenesis and contracted structure; fibrous interruption was shown in the C-P group. The highest percentage of matrix was presented in CB-C-P samples. Collagen and sGAG quantification confirmed that the CB-C-P constructs had statistically higher amounts than the C-P and CB-P groups; statistical differences were also found among the groups in terms of biomechanical properties and gene expression. We concluded that cell bricks-enriched PRP gel sufficiently enhanced the morphological stability of the constructs, maintained chondrocyte phenotypes and favoured chondrogenesis in vivo, which suggests that such an injectable, completely biological system is a suitable cell carrier for cell-based cartilage repair.

Tissue-engineered blood vessels have mainly relied on endothelial cells (ECs), smooth muscle cells (SMCs), and biocompatible materials. However, long-term results have revealed several material-related failures, such as stenosis, thromboembolization, and the risk of infection. Furthermore, SMCs from elderly persons have reduced capacity in proliferation and collagen production. Mesenchymal stem cells (MSCs) have the ability to differentiate into multiple cell lineages, including osteoblasts, chondrocytes, ECs, and SMCs. In the current experiment, rabbit MSCs were cultured to form a cell sheet. A tissue-engineered vascular graft (TEVG) was fabricated by rolling the MSC sheet around a mandrel. The TEVG was implanted into a defect of the common carotid artery after it was examined macroscopically and microscopically. Hematoxylin and eosin staining showed that cell sheet was composed of five to seven layers of cells with the thickness of 40-50 µm. Results from the adhesion assay revealed that MSCs had similar antiplatelet adhesion property to ECs. Histological analysis of TEVGs showed that the layers of the cell sheet had fully fused in vitro. After implantation, TEVGs had excellent patency and integrated well with the native vessel. The structure of the TEVGs was similar to that of the native artery 4 weeks after implantation. Electron microscopy showed that the implanted TEVGs endothelialized. These results indicated that a completely biological TEVG could be assembled with autologous MSCs. These TEVGs are useful for revascularization in humans, which would reduce the occurrence of complications caused by foreign materials.

The most important problem for the survival of thick 3-dimensional tissues is the lack of vascularization in the context of bone tissue engineering. In this study, a modified arteriovenous loop (AVL) was developed to prefabricate an axial vascularized tissue engineering coral bone in rabbit, with comparison of the arteriovenous bundle (AVB) model. An arteriovenous fistula between rabbit femoral artery and vein was anastomosed to form an AVL. It was placed in a circular side groove of the coral block. The complex was wrapped with an expanded-polytetrafluoroethylene membrane and implanted beneath inguinal skin. After 2, 4, 6 and 8 weeks, the degree of vascularization was evaluated by India ink perfusion, histological examination, vascular casts, and scanning electron microscopy images of vascular endangium. Newly formed fibrous tissues and vasculature extended over the surfaces and invaded the interspaces of entire coral block. The new blood vessels robustly sprouted from the AVL. Those invaginated cavities in the vascular endangium from scanning electron microscopy indicated vessel's sprouted pores. Above indexes in AVL model are all superior to that in AVB model, indicating that the modified AVL model could more effectively develop vascularization in larger tissue engineering bone.

Clinical application of platelet-rich plasma (PRP)-based injectable tissue engineering is limited by weak mechanical properties and a rapid fibrinolytic rate. We proposed a new strategy, a cell bricks-stabilized PRP injectable system, to engineer and regenerate cartilage with stable morphology and structure in vivo. Chondrocytes from the auricular cartilage of rabbits were isolated and cultured to form cell bricks (fragmented cell sheet) or cell expansions. Fifteen nude mice were divided evenly (n = 5) into cells-PRP (C-P), cell bricks-PRP (CB-P) and cell bricks-cells-PRP (CB-C-P) groups. Cells, cell bricks or a cell bricks/cells mixture were suspended in PRP and were injected subcutaneously in animals. After 8 weeks, all the constructs were replaced by white resilient tissue; however, specimens from the CB-P and CB-C-P groups were well maintained in shape, while the C-P group appeared distorted, with a compressed outline. Histologically, all groups presented lacuna-like structures, glycosaminoglycan-enriched matrices and positive immunostaining of collagen type II. Different from the uniform structure presented in CB-C-P samples, CB-P presented interrupted, island-like chondrogenesis and contracted structure; fibrous interruption was shown in the C-P group. The highest percentage of matrix was presented in CB-C-P samples. Collagen and sGAG quantification confirmed that the CB-C-P constructs had statistically higher amounts than the C-P and CB-P groups; statistical differences were also found among the groups in terms of biomechanical properties and gene expression. We concluded that cell bricks-enriched PRP gel sufficiently enhanced the morphological stability of the constructs, maintained chondrocyte phenotypes and favoured chondrogenesis in vivo, which suggests that such an injectable, completely biological system is a suitable cell carrier for cell-based cartilage repair.

The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP).We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous β-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model.The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site.The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings.

The most important factor for the survival of thick three-dimensional tissues is the degree of vascularization. In this study, a modified arteriovenous loop (AVL) model was developed to prefabricate an axial vascularized tissue-engineered coral bone. In group A (n = 28), an arteriovenous fistula between rabbit femoral artery and vein was anastomosed to form an AVL. The AVL was placed in a coral block (6 × 8 × 10 mm 3 ) as a vascular carrier. The complex was wrapped with polytetrafluoroethylene membrane and implanted subcutaneously. In group B (n = 20), there was no vascular carrier, and the same dimensional coral was directly implanted beneath inguinal skin. After 2, 4, 6, and 8 weeks, the rabbits were perfused with heparinized saline (for scanning electron microscopy), India ink (for histological examination), and ethylene perchloride (for vascular casts) via the abdominal aorta. In group A, histology showed that newly formed vasculature extended over the surfaces and invaded the entire coral blocks. The vascular density was significantly superior to that in group B. Vascular casts showed that new blood vessels robustly sprouted from the AVL. Scanning electron microscopy demonstrated that there were minute sprouting cavities in the vascular endangium. In this model, an axial vascularized coral bone could be effectively constructed.

The use of exogenous scaffolds to engineer bone tissue faces several drawbacks including insufficient biological activity, potential immunogenicity, elevated inflammatory reaction, fluctuating degradation rate, and low cell-attachment efficiency. To circumvent these limitations, we sought to engineer large scaffold-free bone tissue using cell sheets. We harvested intact cell sheets from bone marrow stromal cells using a continuous culture method and a scraping technique. The cell sheets were then rolled and fabricated into large constructs. Finally, the constructs were implanted into the subcutaneous pockets of nude mice. The cells within the sheet maintained in vitro osteogenic potential after osteoblast differentiation. Computed tomography scans and histological examination confirmed new bone formation in vivo. Additionally, the engineered bone exhibited enhanced compressive strength. Our results indicate that the BMSC sheets can facilitate the formation of functional three-dimensional bone tissue without the use of exogenous scaffolds. Hence, the study provides an intriguing alternative strategy for bone repair.

Due to the limited regenerative capacity of cartilage tissues, articular cartilage defect caused by various lesions remains a problem to be resolved. Tissue engineering provided a valuable alternative to current therapeutic approaches, which is expected to greatly reduce the need of joint replacement. Scaffold, acting as cell carrier, plays an important role in maintaining cells in defect sites, thus facilitates the chondrogenesis. However, an open operation is often needed to implant the cell/scaffold composite, to find a less invasive way to delivering the complex into the defect site would be desirable. Different from synthetic and other nature derived scaffold, platelet-rich plasma (PRP) is a fraction of plasma which contains multiple growth factors and could be clotted when mixed with thrombin. Therefore, we hypothesized that PRP could be used as an autologous cell carrier to inject and fix chondrocytes into the defect site of articular cartilage. With the assistance of arthroscope, the defect could be precisely located, and injectable PRP-Cell composite would make the operation micro-invasive and simple.

This study evaluated the effect of platelet-rich plasma (PRP) on the bone formation of marrow stromal cells (MSCs) in porous coral. MSCs in 50 μl of PRP were seeded into natural coral disks (diameter 8.0 mm; thickness 2.0 mm). The composites were clotted and cultured in vitro or implanted subcutaneously into nude mice. Coral scaffolds loading MSCs or PRP alone acted as control. Alkaline phosphatase (ALP) activity of the specimens cultured in vitro for 7 and 14 days was measured, and the level of ectopic bone formation was investigated 4 and 8 weeks after operation. The samples from the coral/PRP/MSC group exhibited significantly higher ALP activity, compared with that from the coral/MSC group or the coral/PRP group (p

Treatment and management of tracheal defects remain challenges in head and neck surgery. The purposes of this study were to explore a novel strategy to fabricate tissue-engineered trachea by using chondrocyte macroaggregate, and evaluate the feasibility of creating tracheal cartilage equivalents grown in the shape of cylindrical structure without scaffold. Chondrocytes from rabbit cartilage were expanded and seeded into a culture dish at high density to form mechanically stable chondrocyte macroaggregate. Once the chondrocyte macroaggregate was harvested by scrapping technique, it was wrapped around a silicon tube and implanted subcutaneously into the cell donor rabbit. Eight weeks later, specimens were harvested and analyzed for gross appearance, and histological, biochemical, and biomechanical properties. These values were compared with native rabbit cartilage. It was found that expanded chondrocytes could be harvested as a coherent cellular macroaggregate and could be fabricated into a tubelike graft. After in vivo implantation, cartilage-like tissue with cylindrical structure was regenerated successfully. Histological analysis showed engineered trachea cartilage consisted of evenly spaced lacunae embedded in a matrix rich in proteoglycans; type II collagen was also highly expressed in this engineered trachea cartilage. In a conclusion, based on the chondrocyte macroaggregate strategy, tracheal cartilage equivalents with cylindrical shape could be successfully reconstructed. This construct has advantages of high cell-seeding efficiency, good nutritional perfusion, and minimal inflammatory reaction, which provided a highly effective cartilage graft substitute and could be useful in many situations of trachea-cartilage loss encountered in clinical practice.

In this study, cell sheets comprising multilayered porcine bone marrow stromal cells (BMSC) were assembled with fully interconnected scaffolds made from medical-grade polycaprolactone-calcium phosphate (mPCL-CaP), for the engineering of structural and functional bone grafts. The BMSC sheets were harvested from culture flasks and wrapped around pre-seeded composite scaffolds. The layered cell sheets integrated well with the scaffold/cell construct and remained viable, with mineralized nodules visible both inside and outside the scaffold for up to 8 weeks culture. Cells within the constructs underwent classical in vitro osteogenic differentiation with the associated elevation of alkaline phosphatase activity and bone-related protein expression. In vivo, two sets of cell-sheet-scaffold/cell constructs were transplanted under the skin of nude rats. The first set of constructs (5 x 5 x 4mm(3)) were assembled with BMSC sheets and cultured for 8 weeks before implantation. The second set of constructs (10 x 10 x 4mm(3)) was implanted immediately after assembly with BMSC sheets, with no further in vitro culture. For both groups, neo cortical and well-vascularised cancellous bone were formed within the constructs with up to 40% bone volume. Histological and immunohistochemical examination revealed that neo bone tissue formed from the pool of seeded BMSC and the bone formation followed predominantly an endochondral pathway, with woven bone matrix subsequently maturing into fully mineralized compact bone; exhibiting the histological markers of native bone. These findings demonstrate that large bone tissues similar to native bone can be regenerated utilizing BMSC sheet techniques in conjunction with composite scaffolds whose structures are optimized from a mechanical, nutrient transport and vascularization perspective.

Bone regeneration is often needed for many aesthetic and reconstructive procedures. Tissue engineering provided a promising approach to supplement existing treatment strategies. In this study, we aimed to evaluate the effect of reconstructing mandibular defect by using bioceramics seeded with bone marrow derived osteoblasts.Canine's autologous marrow stromal cells were Culture-expanded and induced to osteoblastic phenotype, then were seeded into prepared porous beta-tricalcium phosphate, after being incubated in vitro. The cell/ scaffold complexes were implanted into the prepared defect in canines' mandibula and fixed by internal rigid fixation. In control groups, beta-tricalcium phosphate alone and autologous iliums were implanted into the prepared defects. Twelve weeks after implantation, the specimens were examined macroscopically and histologically.In experimental group and autologous iliums group, new bone grafts were successfully developed at 12 weeks after implantation and repaired the continuity of the mandibula. Histologically, newly formed bone could be observed on the surface and in the pores of beta-tricalcium phosphate in the cell/scaffold group, whereas incomplete bone repair was found in pure beta-tricalcium phosphate group.The harvested bone marrow derived osteoblasts possess the ability to form new bone tissue when seeded onto porous beta-tricalcium phosphate, which shows the potential of using this method to repair large segmental mandibular defect clinically.

The cuttlebones, harvested from cuttles, undergo the chemical reaction in high temperature and high pressure for a certain time. The products are qualitatively analysed, and spacial structure observation and cytocompatibility are tested. The results show that the chemical component of the cuttlebone is CaCO3 and the crystal type is aragonite. Cuttlebones undergo a hydro-thermal reaction, and thus transform into hydroxyapatite-that is, the cuttlebone-transformed hydroxyapatite(CBHA). The CBHA materials have the interconnected microporous network structures. Under the high magnification, CBHAs appear to have many micro-spheres, thus construct a new self-assembled nano-material system. The marrow stromal osteoblasts can adhere to and proliferate well on the surface of the CBHAs. These results show that CBHAs have good biocompatibility. Therefore, it can be a potential candidate scaffold for bone tissue engineering.

Purpose Platelet-rich plasma (PRP) has been widely applied to promote tissue healing and used as a novel injectable scaffold in bone tissue engineering. However, there is no report about its feasibility to support chondrogenesis. This study aimed to investigate the feasibility of a PRP carrier to deliver chondrocytes and regenerate cartilage tissues in a rabbit model via injection. Materials and Methods Eight New Zealand rabbits were divided into a chondrocytes/PRP group (n = 4) and a PRP-alone group (n = 4). Chondrocytes harvested from the auricular root of New Zealand rabbits were cultured and harvested. The chondrocytes were then mixed with PRP solution to generate chondrocytes/PRP composites with final cellular density of 5.0 × 107/mL. Bovine thrombin was used as a cross-linking agent to gel chondrocytes/PRP composites, then, the composites were injected subcutaneously into the dorsal tissue of cell donor animals. As controls, PRP alone was injected into another 4 rabbits. At the second month after injection, rabbits were prepared for magnetic resonance imaging. The samples were then harvested for macroscopical examination, histological analysis, and glycosaminoglycan quantification. Results Two months after injection, the hard knobbles were easily palpated under the dorsal skin of the animals in the chondrocytes/PRP group, and magnetic resonance images showed the presence of cartilage-like tissues. In histological analysis, formation of new cartilage was observed in the chondrocytes/PRP composites. Safranin-O staining and Masson’s trichrome staining showed proteoglycan and collagen were produced in matrices. In contrast, no tissue formed in the PRP-alone group. Conclusions This study suggests the feasibility of using PRP as injectable scaffold seeded with chondrocytes to regenerate cartilage and showed the potential of using this method for the reconstruction of cartilage defects.

To evaluate repair of critical-sized cranial defect with tissue engineered bone fabricated by coral, bone mesenchymal stem cells (MSCs) and sustainedly released recombinant human bone morphogenetic protein 2 (rhBMP-2) by collagen.Three scaffolds of rhBMP-2 + coral, collagen + rhBMP-2 + coral and MSCs+collagen+rhBMP-2+coral were fabricated. Forty New Zealand rabbits were made the models of critical-sized defects and divided into 5 groups according to different implants: group I, auto-ilium; group II, coral; group III, rhBMP-2+coral; group IV, collagen+rhBMP-2+coral; and group V , MSCs+collagen+rhBMP-2+coral. Repair of bone defect was evaluated after 8 and 16 weeks of implantation by gross observation, X-ray, HE staining and Masson's trichrome staining.Repair of bone defect in group V was similar to that in group I, and was better than that in group IV; and group III was worse. The gross appearance showed that defect region filled with bony tissue which had similar strength to adjacent bone and formed bone union with surrounding bone. The X-ray result displayed high radiopacity (80.45% +/- 2.52% in the 16th week). Histological observation showed new lamellar bone tissue and with few pore blank area. However, only transparent fibrous tissue filled the defect in group II.Collagen may be a suitable sustained release system for rhBMP-2. And MSCs may have important effect on enhancing repair of bone defect. Tissue engineered bone fabricated by MSCs+collagen+rhBMP-2+coral may be a useful material for bone defect repair.

Purpose 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. Materials and methods 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 × 10 8 /mL in 200 μL 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. Results 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. Conclusion 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.

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.

Seed cell study is an essential area in the research of tissue engineering. To evaluate the potentiality of marrow stromal cell(MSCs) as seed cell in the regeneration of tissue engineered cartilage, formation of cartilage nodules by culture expanded MSCs pellets under the induction of TGF-beta was investigated.MSCs were cultured and expanded in vitro. Cell pellets containing 1 x 10(6) MSCs were obtained by centrifuging MSCs solution at 1,000 r/min in 5 ml centrifugation tube. Pellets were exposed to cell culture media containing 20 ng/ml TGF-beta for 7 days and then cultured for another 7 and 21 days. The nodules were moved out of the tube and cartilage formation was observed by stereomicroscope, light microscope and electronic microscope.10 days after exposure to TGF-beta, pellets contracted and formed small and round nodules on the bottom of the tubes. The nodules grew bigger slowly and reached maximal diameter of 1.8 mm in 28 days. The surface of the nodules was smooth and bright white. Histological examination showed that extra cellular matrix formed in 14 days and in some areas cells situated in lacuna. In 28 days' specimens, a lot of cells situated in lacuna could be observed and the histological appearance looked much similar to cartilage. Electronic microscope observation demonstrated that in 28 days' specimens a large amount of collagen fiber existed.Under the induction of TGF-beta, MSCs could differentiate into chondrogenesis cell and form cartilaginous nodules in vitro. This indicated that MSCs could be the potential seed cells in the regeneration of cartilage employing method of tissue engineering.

To investigate the feasibility of using marrow stromal osteoblast (MSO) as bone derived cell and using cancellous bone matrix (CBM) as scaffold for bone tissue engineering, the subcutaneous osteogenesis of MSO-CBM compound artificial bone (MCCAB) was observed in the experiment.The marrow stromal cells of adult New Zealand rabbits cultivated and induced in vitro were used to form MCCAB by mixing, seeding and solidifying methods assisted by alginate. The MCCABs were auto-transplanted subcutaneously into the rabbits for 4 to 8 weeks. The alginate-cancellous bone matrix composites or the cancellous bone matrix alone were implanted as control. The effectiveness of bone formation was assessed by means of roentgenography, histology and computerized histomorphometry.The osteogenesis of MCCABs was better than that of the alginate-cancellous bone matrix composites and of the cancellous bone matrixes. In the MCCABs, both intramembranous and cartilaginous osteogeneses were seen but the former was obvious. In the control, only slight cartilaginous osteogeneses were seen.The osteogeneses of the MCCABs constructed by using tissue engineering method were obvious when transplanted subcutaneously. The MSO and CBM can be used as good bone-derived cell and scaffold material respectively for tissue-engineered bone construction.