Engineering tubular bone constructs

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.     

A cell leakproof PLGA‐collagen hybrid scaffold for cartilage tissue engineering

A cell leakproof porous poly(DL ‐lactic‐co‐glycolic acid) (PLGA)‐collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi‐layered PLGA mesh. The PLGA‐collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a bi‐layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi‐layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immunostaining demonstrated that type II collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real‐time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage‐like tissue when being cultured in the cell leakproof PLGA‐collagen hybrid scaffold. The cell leakproof PLGA‐collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Application of a cell sheet–polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability

We have succeeded in fabricating a cell sheet–polymer film complex involving a temperature‐sensitive polymer that has enough mechanical strength that can be manipulated even by forceps. The polymer film can be removed by lowering the temperature after transplantation, demonstrating its potential use in regenerative medicine. Recently, tissue engineering involving cell sheets was developed, tissues being fabricated by layering of these cell sheets. This technique promises high density cell packing, which is important for native cell functions, and successful heart therapy using cardiac cell sheets has been reported. On the other hand, the fabrication of a large tissue using cell sheets is difficult because of fragility of the cell sheets. Here, we have developed a novel method in which cells are attached to a temperature‐sensitive poly‐N‐isopropylacrylamide film mixed with laminin and collagen IV, and report that the cell sheet–polymer film complex can be manipulated with forceps. A cell sheet can be removed from the polymer film by lowering the temperature after the manipulation. We have utilized this technique for the primary myocardium and fabricated a physiologically active multi‐layered cardiac cell sheet. By applying a micropattern to this polymer film, we have succeeded in making a skeletal muscle cell sheet in which myotubes are oriented in the desired direction. Overall, we showed that this method is useful for cell sheet manipulation, morphogenesis, and transplantation. Biotechnol. Bioeng. 2009;103: 370–377. © 2009 Wiley Periodicals, Inc.     

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration.     

Responsive systems for cell sheet detachment

Cell sheet engineering has been progressing rapidly during the past few years and has emerged as a novel approach for cell based therapy. Cell sheet harvest technology enables fabrication of viable, transplantable cell sheets for various tissue engineering applications. Currently, the majority of cell sheet studies use thermo-responsive systems for cell sheet detachment. However, other responsive systems began showing their potentials for cell sheet harvest. This review provides an overview of current techniques in creating cell sheets using different types of responsive systems including thermo-responsive, electro-responsive, photo-responsive, pH-responsive and magnetic systems. Their mechanism, approach, as well as applications for cell detachment have been introduced. Further development of these responsive systems will allow efficient cell sheet harvesting and patterning of cells to reconstruct complex tissue for broad clinical applications.     

Influence of collagen and chondroitin sulfate (CS) coatings on poly-(lactide-co-glycolide) (PLGA) on MG 63 osteoblast-like cells

Poly-(lactide-co-glycolide) (PLGA) is an FDA-approved biodegradable polymer which has been widely used as a scaffold for tissue engineering applications. Collagen has been used as a coating material for bone contact materials, but relatively little interest has focused on biomimetic coating of PLGA with extracellular matrix components such as collagen and the glycosaminoglycan chondroitin sulfate (CS). In this study, PLGA films were coated with collagen type I or collagen I with CS (collagen I/CS) to investigate the effect of CS on the behaviour of the osteoblastic cell line MG 63. Collagen I/CS coatings promoted a significant increase in cell number after 3 days (in comparison to PLGA) and after 7 days (in comparison to PLGA and collagen-coated PLGA). No influence of collagen I or collagen I/CS coatings on the spreading area after 1 day of culture was observed. However, the cells on collagen I/CS formed numerous filopodia and displayed well developed vinculin-containing focal adhesion plaques. Moreover, these cells contained a significantly higher concentration of osteocalcin, measured per mg of protein, than the cells on the pure collagen coating. Thus, it can be concluded that collagen I/CS coatings promote MG 63 cell proliferation, improve cell adhesion and enhance osteogenic cell differentiation.     

Electrospun PLGA�Csilk fibroin�Ccollagen nanofibrous scaffolds for nerve tissue engineering

Electrospun nanofibrous scaffolds varying different materials are fabricated for tissue engineering. PLGA, silk fibroin, and collagen-derived scaffolds have been proved on good biocompatibility with neurons. However, no systematic studies have been performed to examine the PLGA-silk fibroin-collagen (PLGA-SF-COL) biocomposite fiber matrices for nerve tissue engineering. In this study, different weight ratio PLGA-SF-COL (50:25:25, 30:35:35) scaffolds were produced via electrospinning. The physical and mechanical properties were tested. The average fiber diameter ranged from 280 + 26 to 168 + 21 nm with high porosity and hydrophilicity; the tensile strength was 1.76 ± 0.32 and 1.25 ± 0.20 Mpa, respectively. The results demonstrated that electrospinning polymer blending is a simple and effective approach for fabricating novel biocomposite nanofibrous scaffolds. The properties of the scaffolds can be strongly influenced by the concentration of collagen and silk fibroin in the biocomposite. To assay the cytocompatibility, Schwann cells were seeded on the scaffolds; cell attachment, growth morphology, and proliferation were studied. SEM and MTT results confirmed that PLGA-SF-COL scaffolds particularly the one that contains 50% PLGA, 25% silk fibroin, and 25% collagen is more suitable for nerve tissue engineering compared to PLGA nanofibrous scaffolds.     

The use of adipose-derived stem cells as sheets for wound healing

Cellular therapies have shown immense promise in the treatment of nonhealing wounds. Cell sheets are an emerging strategy in tissue engineering, and these cell sheets are promising as a delivery method of mesenchymal stem cells to the wound bed. Cell sheet technology utilizes temperature dependent polymers to allow for lifting of cultured cells and extracellular matrix without the use of digestive enzymes. While mesenchymal stem cells (MSCs) have shown success in cell sheets for myocardial repair, examination of cell sheets in the field of wound healing has been limited. We previously developed a novel cell sheet composed of human adipose-derived stem cells (ASCs). Both single and triple layer cell sheets were examined in a full-thickness murine wound model. The treatment cell sheets were compared with untreated controls and analyzed at timepoints of 7, 14, 18 and 21 d. The ASC cell sheets showed increased healing at 7, 14 and 18 d, and this effect was increased in the triple layer cell sheet group. Future development of these cell sheets will focus on increasing angiogenesis in the wound bed, utilizing multiple cell types, and examining allogeneic cell sheets. Here we review our experiment, expand upon our future directions and discuss the potential of an off-the-shelf cell sheet. In the field of wound healing, such a cell sheet is both clinically and scientifically exciting.     

Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels

In this study, a novel yet simple method, using a thermoreversible hydrogel system coated on tissue culture polystyrene (TCPS) dishes, was developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20 degrees C. For the applications to cell culture, only those aqueous MC compositions that may form a gel at 37 degrees C were chosen for the study. It was found that the hydrogel coating composed of 8% MC blended with 10 g/L PBS (phosphate buffered saline) (the MC/PBS hydrogel, with a gelation temperature of approximately 25 degrees C) stayed intact throughout the entire course of cell culture. To improve cell attachments, the MC/PBS hydrogel at 37 degrees C was evenly spread with a neutral aqueous collagen at 4 degrees C. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet formed on the surface of the MC/PBS/collagen hydrogel. When the grown cell sheet was placed outside of the incubator at 20 degrees C, it detached gradually from the surface of the thermoreversible hydrogel spontaneously, without treating with any enzymes. The results obtained in the MTT assay demonstrated that the cells cultured on the surface of the MC/PBS/collagen hydrogel had an even better activity than those cultured on an uncoated TCPS dish. After harvesting the detached cell sheet, the remaining viscous hydrogel system is reusable. Additionally, the developed hydrogel system can be used for culturing a multilayer cell sheet. The obtained living cell sheets may be used for tissue reconstructions.     

Adenoviral BMP-2 gene transfer in mesenchymal stem cells: in vitro and in vivo bone formation on biodegradable polymer scaffolds

The aim of this study was to determine the feasibility of adenoviral gene transfer into primary human bone marrow osteoprogenitor cells in combination with biodegradeable scaffolds to tissue-engineer bone. Osteoprogenitors were infected with AxCAOBMP-2, a vector carrying the human BMP-2 gene. Alkaline phosphatase activity was induced in C2C12 cells following culture with conditioned media from BMP-2 expressing cells, confirming successful secretion of active BMP-2. Expression of alkaline phosphatase activity, type I collagen and mineralisation confirmed bone cell differentiation and maintenance of the osteoblast phenotype in extended culture for up to 6 weeks on PLGA porous scaffolds. In vivo implantation of adenoviral osteoprogenitor constructs on PLGA biodegradeable scaffolds, using diffusion chambers, also demonstrated bone cell differentiation and production of bone tissue. The maintenance of the osteoblast phenotype in extended culture and generation of mineralised 3-D scaffolds containing such constructs indicate the potential of such bone tissue engineering approaches in bone repair.     

Repair of rabbit ulna segmental bone defect using freshly isolated adipose-derived stromal vascular fraction

Background aimsStromal vascular fractions (SVF) from adipose tissue have heterogeneous cell populations, and include multipotent adipose-derived stem cells. The advantages of using of SVF include the avoidance of an additional culture period, a reduced risk of extensive cell contamination, and cost-effectiveness.MethodsUnilateral 20-mm mid-diaphyseal segmental defects in rabbit ulna were treated with one of the following: polylactic glycolic acid (PLGA) scaffold alone (group 1, control), a PLGA scaffold with undifferentiated SVF cells (group 2), or a PLGA scaffold with osteogenically differentiated SVF cells (group 3). At 8 weeks after implantation, five rabbits in each treatment group were killed to assess bone defect healing by plain radiography, quantitative microcomputed tomography and histology.ResultsThe SVF cells were well grown on PLGA scaffolds and expressed type I collagen and alkaline phosphatase (ALP). The intensity of ALP and OPN gene expressions in osteogenic medium culture were increased from 14 days to 28 days. In vivo evaluations at 8 weeks showed that treatment of SVF cells with or without osteogenic differentiation resulted in more bone formation in the critically sized segmental defects than PLGA scaffold alone. Osteogenically differentiated SVF cells significantly enhanced bone healing compared with undifferentiated SVF cells.ConclusionsAdipose-derived stromal SVF showed osteogenic potential in vitro. Accordingly, SVF could provide a cell source for bone tissue engineering. However, treatment with uncultured SVF cells on bone healing was not satisfactory in the in vivo animal model.     

Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineering

Bone tissue engineering is an interdisciplinary field involving both engineers and cell biologists, whose main purpose is to repair bone anatomical defects and maintain its functions. A novel system that integrates pulsed electromagnetic fields (PEMFs) and bioreactors was applied to bone tissue engineering for regulating osteoblast proliferation and differentiation in'vitro. Osteoblasts were acquired from the calvaria of newborn Wistar rats and isolated after sequential digestion. Poly(DL-lactic-co-glycolic acid) (PLGA) scaffolds were made by the solvent merging/particulate leaching method. Osteoblasts were seeded into porous PLGA scaffolds with 85% porosity and cultured in bioreactors for the 18-day culture period. Cells were exposed to PEMF pulsed stimulation with average (rms) amplitudes of either 0.13, 0.24, or 0.32 mT amplitude. The resulting induced electric field waveform consisted of single, narrow 300 micros quasi-rectangular pulses with a repetition rate of 7.5'Hz. The results showed that PEMF stimulation for 2 and 8 h at .13 mT increased the cell number on days 6 and 12, followed by a decrease on day 18 using 8 h stimulation. However, ALP activity was decreased and then increased on days 12 and 18, respectively. On the other hand, PEMF-treated groups (irrespective of the stimulation time) at 0.32 mT inhibited cell proliferation but enhanced ALP activity during the culture period. These findings suggested that PEMF stimulation with specific parameters had an effect on regulating the osteoblast proliferation and differentiation. This novel integrated system may have potential in bone tissue engineering.     

Technique to accurately quantify collagen content in hyperconfluent cell culture

Tissue engineering aims to regenerate tissues that can successfully take over the functions of the native tissue when it is damaged or diseased. In most tissues, collagen makes up the bulk component of the extracellular matrix, thus, there is great emphasis on its accurate quantification in tissue engineering. It has already been reported that pepsin digestion is able to solubilize the collagen deposited within the cell layer for accurate quantification of collagen content in cultures, but this method has drawbacks when cultured cells are hyperconfluent. In this condition, Pepsin digestion will result in fragments of the cell layers that cannot be completely resolved. These fragments of the undigested cell sheet are visible to the naked eye, which can bias the final results. To the best of our knowledge, there has been no reported method to accurately quantify the collagen content in hyperconfluent cell sheet. Therefore, this study aims to illustrate that sonication is able to aid pepsin digestion of hyperconfluent cell layers of fibroblasts and bone marrow mesenchymal stem cells, to solubilize all the collagen for accurate quantification purposes.     

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 μM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.     

Fabrication of core-sheath structured fibers for model drug release and tissue engineering by emulsion electrospinning

A nanofibrous core-sheath structured scaffold incorporated with bioactive agents is supposed to promote cell migration, proliferation, and gene expressions through the controllable and sustainable release of bioactive agents from the fibers and the preservation of bioactivity. Here we present a novel and effective emulsion electrospinning method for obtaining fluorescein isothiocyanate-dextran (FITC-dextran)/poly(lactic-co-glycolic acid) (PLGA) and type I collagen/PLGA fibrous composite scaffolds. Core-sheath structured fibers with average diameters of 665 nm for FITC-dextran/PLGA and 567 nm for collagen/PLGA were successfully fabricated. In vitro-release profile shows sustained release of encapsulated FITC-dextran from FITC-dextran/PLGA fibers for as long as 7 weeks. The osteoblastic activity of the collagen/PLGA nanofibrous scaffold was investigated employing the osteoblastic-like MC3T3-E1 cell line. The results of the lactate dehydrogenase assay suggested excellent cytocompatibility. Cell proliferation and alkaline phosphatase activity were also ameliorated on this emulsion-electrospun collagen/PLGA fibrous scaffold. All the results indicated that this composite scaffold could support the early stages of osteoblast behavior as well as the immediate/late stages. The emulsion electrospinning process has potential for application in drug-release devices and as a 3-D scaffold in bone regeneration.     

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces to biological hydrogels for rapid and efficient cell micropatterning

Cell transfer printing from patterned poly(ethylene glycol)-oleyl surfaces onto biological hydrogel sheets is investigated herein, as a new cell stamping method for both cell microarray and tissue engineering. By overlaying a hydrogel sheet on the cells immobilized on the poly(ethylene glycol)-oleyl surface and successively peeling it off, the immobilized cells were transferred onto a hydrogel sheet because the adhesive interaction between the cells and the hydrogel was stronger than that between the cells and the poly(ethylene glycol)-oleyl surface. Four types of human cell could be efficiently transferred onto a rigid collagen sheet. The transfer printing ratios, for all cells, were above 80% and achieved within 90 min. A cell microarray was successfully prepared on a collagen gel sheet using the present stamping method. We have also demonstrated that the transferred pattern of endothelial cells is transformed to the patterned tube-like structure on the reconstituted basement membrane matrix. Finally, the patterns of two types of endothelial cell are shown to be easily prepared on the matrix, and the desired tube-like structures, including the orderly pattern of the two different cells, were formed spontaneously. Thus, the present poly(ethylene glycol)-oleyl coated substrates are useful for rapid and efficient cell stamping, in the preparation of multi-cellular pattern on extracellular matrices.     

Effect of different cell sheet ECM microenvironment on the formation of vascular network

The repair and reconstruction of large bone defects remains as a significant clinical challenge mainly due to the insufficient vascularization. The prefabrication of vascular network based on cell sheet technique brings a promising potential for sufficient vascularization due to rich extracellular matrix (ECM) of cell sheets. However, the effect of different cell sheet ECM micro-environment on the formation of a vascular network has not been well understood. Here our goal is to study the effect of different cell sheets on the formation of a vascular network. First we cultured human bone marrow mesenchymal stem cells (hBMSCs) under two culture conditions to obtain osteogenic differentiated cell sheet (ODCS) and undifferentiated cell sheet (UDCS), respectively. Then the human umbilical vein endothelial cells (HUVECs) were seeded onto the surface of the two sheets at different seeding densities to fabricate pre-vascularized cell sheets. Our results indicated that the two sheets facilitated the alignment of HUVECs and promoted the formation of vascular networks. Quantitative analysis showed that the number of networks in ODCS was higher than that in the UDCS. The ECM of the two sheets was remodeled and rearranged during the tubulogenesis process. Furthermore, results showed that the optimal seeding density of HUVECs was 5 × 10⁴ cell/cm². In summary, these results suggest that the vascularized ODCS has a promising potential to construct pre-vascularized tissue for bone repair.     

Time Course of Cell Sheet Adhesion to Porcine Heart Tissue after Transplantation

Multilayered cell sheets have been produced from bone marrow-derived mesenchymal stem cells (MSCs) for investigating their adhesion properties onto native porcine heart tissue. Once MSCs reached confluence after a 7-day culture on a temperature-responsive culture dish, a MSCs monolayer spontaneously detached itself from the dish, when the culture temperature was reduced from 37 to 20°C. The basal extracellular matrix (ECM) proteins of the single cell sheet are preserved, because this technique requires no proteolytic enzymes for harvesting cell sheet, which become a basic building block for assembling a multilayer cell sheet. The thickness of multilayered cell sheets made from three MSC sheets was found to be approximately 60 μm. For investigating the adhesion properties of the basal and apical sides, the multilayered cell sheets were transplanted onto the surface of the heart’s left ventricle. Multilayered cell sheets were histological investigated at 15, 30, 45 and 60 minutes after transplantation by hematoxylin eosin (HE) and azan dyes to determine required time for the adhesion of the multilayered sheets following cell-sheet transplantation. The results showed that only the basal side of multilayered cell sheets significantly enhanced the sheets adhesion onto the surface of heart 30 minutes after transplantation. This study concluded that (1) cell sheets had to be transplanted with its basal side onto the surface of heart tissue and (2) at least 30 minutes were necessary for obtaining the histological adhesion of the sheets to the heart tissue. This study provided clinical evidence and parameters for the successful application of MSC sheets to the myocardium and allowed cell sheet technology to be adapted clinical cell-therapy for myocardial diseases.     

Fabrication of functional three-dimensional tissues by stacking cell sheets in vitro

The fabrication of 3D tissues retaining the original functions of tissues/organs in vitro is crucial for optimal tissue engineering and regenerative medicine. The fabrication of 3D tissues also contributes to the establishment of in vitro tissue/organ models for drug screening. Our laboratory has developed a fabrication system for functional 3D tissues by stacking cell sheets of confluent cultured cells detached from a temperature-responsive culture dish. Here we describe the protocols for the fabrication of 3D tissues by cell sheet engineering. Three-dimensional cardiac tissues fabricated by stacking cardiac cell sheets pulsate spontaneously, synchronously and macroscopically. Via this protocol, it is also possible to fabricate other tissues, such as 3D tissue including capillary-like prevascular networks, from endothelial cells sandwiched between layered cell sheets. Cell sheet stacking technology promises to provide in vitro tissue/organ models and more effective therapies for curing tissue/organ failures.     

Fabrication of collagen hybridized elastic PLCL for tissue engineering

Biodegradable elastic poly(l-lactide-co-ε-caprolactone) (PLCL) (50:50) copolymer was blended with collagen (0.05, 0.1 and 0.2% w/w) in an acidic dioxane solution to form a collagen/PLCL hybrid material suitable for tissue engineering applications. Stability and dispersivity of collagen on collagen/PLCL hybrid films and collagen coated PLCL films under mechanical stress were determined by a collagen release test and water contact angle measurement. Hybrid films had a higher stability than collagen-coated PLCL films. Elastic recovery as well as high interconnectivity and uniform pore morphology of the hybrid scaffolds were not affected by the collagen concentration. Fibroblasts (NIH-3T3) cell culture test was performed for cell growth and viability evaluation. Collagen concentration had little affect on the initial cell adhesion after 4 h cell culture; but after 48 h cell culture, increased cell proliferation on the hybrid films was observed. The hybrid material can be applied as a scaffold for vessel and cartilage regeneration for mechano-active tissue engineering.