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Bioreactors for tissue engineering.

Bioreactors are essential in tissue engineering, not only because they provide an in vitro environment mimicking in vivo conditions for the growth of tissue substitutes, but also because they enable systematic studies of the responses of living tissues to various mechanical and biochemical cues. The basic principles of bioreactor design are reviewed, the bioreactors commonly used for the tissue engineering of cartilage, bone and cardiovascular systems are assessed in terms of their performance and usefulness. Several novel bioreactor types are also reviewed.

Animals↗

Computer-aided tissue engineering: overview, scope and challenges.

Advances in computer-aided technology and its application with biology, engineering and information science to tissue engineering have evolved a new field of computer-aided tissue engineering (CATE). This emerging field encompasses computer-aided design (CAD), image processing, manufacturing and solid free-form fabrication (SFF) for modelling, designing, simulation and manufacturing of biological tissue and organ substitutes. The present Review describes some salient advances in this field, particularly in computer-aided tissue modeling, computer-aided tissue informatics and computer-aided tissue scaffold design and fabrication. Methodologies of development of CATE modelling from high-resolution non-invasive imaging and image-based three-dimensional reconstruction, and various reconstructive techniques for CAD-based tissue modelling generation will be described. The latest development in SFF to tissue engineering and a framework of bio-blueprint modelling for three-dimensional cell and organ printing will also be introduced.

Animals↗

Flap prefabrication and prelamination with tissue-engineered cartilage.

In reconstructive surgery, the integration of tissue-engineered cartilage in a prefabricated free flap may make it possible to generate flaps combining a variety of tissue components, to meet the special requirements of particular defects. One aim of the present study was to investigate prefabrication of a microvascular free flap by implanting a vessel loop under a skin flap in a rabbit model. A second aim was to report on the authors' preliminary experiences in prelaminating prefabricated flaps with autologous tissue-engineered cartilage, in terms of matrix development, inflammatory reaction, and host-tissue interaction. The flap was prefabricated by implanting a vessel loop under a random-pattern abdominal skin flap. The tissue-engineered cartilage constructs were made by isolating chondrocytes from auricular biopsies. Following a period of amplification, the cells were seeded onto a non-woven scaffold made of a hyaluronic-acid derivative and cultivated for 2 weeks. One cell-biomaterial construct was placed beneath the prefabicated flap, and two additional constructs were placed subcutaneously and intramuscularly. In addition, a biomaterial sample without cells was placed subcutaneously to provide a control. All implanted specimens were left in position for 6 or 12 weeks. Neovascularization in the prefabricated flap and biomaterial construct was analyzed by angiography. After explantation, the specimens were examined by histologic and immunohistochemical methods. The prefabricated flaps showed a well-developed network of blood vessels between the implanted vessel loop and the original random-pattern blood supply. The tissue-engineered constructs remained stable in size and showed signs of tissue similar to hyaline cartilage, as evidenced by the expression of cartilage-specific collagen type II and proteoglycans. No inflammatory reactions were observed. The physiologic environment of the autologous rabbit model provided favorable conditions for matrix deposition and maturation of the cell-biomaterial constructs. These initial results demonstrated the potential of prefabricating an axial perfused flap, combined with tissue-engineered cartilage, thus creating functionally competent tissue components for reconstructive surgery with minimal donor-site morbidity.

Angiography↗

Approaches to improve angiogenesis in tissue-engineered skin.

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

Cell Culture Techniques↗

Biological and biophysical principles in extracorporal bone tissue engineering. Part I.

Advances in the field of bone tissue engineering have encouraged physicians to introduce these techniques into clinical practice. Bone tissue engineering is the construction, repair or replacement of damaged or missing bone in humans or animals. Engineering of bone can take place within the animal body or extracorporal in a bioreactor for later grafting into the body. Appropriate cell types and non-living substrata are minimal requirements for an extracorporal tissue engineering approach. This review discusses the biological and biophysical background of in vitro bone tissue engineering. Biochemical and biophysical stimuli of cell growth and differentiation are regarded as potent tools to improve bone formation in vitro. The paper focuses on basic principles in extracorporal engineering of bone-like tissues, intended to be implanted in animal experiments and clinical studies. Particular attention is given in this part to the contributions of cell and material science to the development of bone-like tissues. Several approaches are at the level of clinical applicability and it can be expected that widespread use of engineered bone constructs will change the surgeon's work in the near future.

Animals↗

Experimental and clinical experience with tissue engineering techniques for urethral reconstruction.

Tissue engineering has been proposed as a strategy for urethral reconstruction. This may involve matrices alone, wherein the body's natural ability to regenerate is used to orient or direct new tissue growth, or the use of matrices with cells. Acellular collagen matrices derived from donor bladder submucosa have been used both experimentally and clinically for onlay urethral replacement with good success at our center. If a tubularized urethral repair is needed, the use of cells on the collagen matrix is essential for adequate tissue formation. Tissue engineering techniques are useful for urethral reconstruction.

Biocompatible Materials↗

Engineering controllable anisotropy in electrospun biodegradable nanofibrous scaffolds for musculoskeletal tissue engineering.

Many musculoskeletal tissues exhibit significant anisotropic mechanical properties reflective of a highly oriented underlying extracellular matrix. For tissue engineering, recreating this organization of the native tissue remains a challenge. To address this issue, this study explored the fabrication of biodegradable nanofibrous scaffolds composed of aligned fibers via electrospinning onto a rotating target, and characterized their mechanical anisotropy as a function of the production parameters. The characterization showed that nanofiber organization was dependent on the rotation speed of the target; randomly oriented fibers (33% fiber alignment) were produced on a stationary shaft, whereas highly oriented fibers (94% fiber alignment) were produced when rotation speed was increased to 9.3m/s. Non-aligned scaffolds had an isotropic tensile modulus of 2.1+/-0.4MPa, compared to highly anisotropic scaffolds whose modulus was 11.6+/-3.1MPa in the presumed fiber direction, suggesting that fiber alignment has a profound effect on the mechanical properties of scaffolds. Mechanical anisotropy was most pronounced at higher rotation speeds, with a greater than 33-fold enhancement of the Young's modulus in the fiber direction compared to perpendicular to the fiber direction when the rotation speed reached 8m/s. In cell culture, both the organization of actin filaments of human mesenchymal stem cells and the cellular alignment of meniscal fibroblasts were dictated by the prevailing nanofiber orientation. This study demonstrates that controllable and anisotropic mechanical properties of nanofibrous scaffolds can be achieved by dictating nanofiber organization through intelligent scaffold design.

Absorbable Implants↗

An overview of tissue engineered bone.

Numerous important developments in tissue engineering of new bone during the last 10 years are reviewed. Early efforts to combine cells with biocompatible materials are described and applications of this technology are presented with particular focus on uses in orthopaedics and maxillofacial surgery. Basic principles of tissue engineering focusing on cell biology and materials science as used currently in the field are presented. Finally, future challenges are outlined from the perspective of integrating technologies from medicine, biology, and engineering in hopes of translating tissue engineering to clinical applications.

Animals↗

Cartilage tissue engineering with silk scaffolds and human articular chondrocytes.

Adult cartilage tissue has poor capability of self-repair, especially in case of severe cartilage damage due to trauma or age-related degeneration. Autologous cell-based tissue engineering using three-dimensional (3-D) porous scaffolds has provided an option for the repair of full thickness defects in adult cartilage tissue. Mesenchymal stem cells (MSCs) and chondrocytes are the two major cell sources for cartilage tissue engineering. Silk fibroin as a naturally occurring degradable fibrous protein with unique mechanical properties, excellent biocompatibility and process-ability has demonstrated strong potential for skeletal tissue engineering. The present study combined adult human chondrocytes (hCHs) with aqueous-derived porous silk fibroin scaffolds for in vitro cartilage tissue engineering. The results were compared with a previous study using the same scaffolds but using MSCs to generate the cartilage tissue outcomes. Culture-expanded hCHs attached to, proliferated and re-differentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-beta1, based on cell morphology, levels of cartilage-related gene transcripts, and the presence of a cartilage-specific ECM. Cell density was critical for the redifferentiation of culture-expanded hCHs in the 3-D aqueous-derived silk fibroin scaffolds. The level of cartilage-related transcripts (AGC, Col-II, Sox 9 and Col-II/Col-I ratio) and the deposition of cartilage-specific ECM were significantly upregulated in constructs initiated with higher seeding density. The hCH-based constructs were significantly different than those formed from MSC-based constructs with respect to cell morphology, zonal structure and initial seeding density needed to successfully generate engineered cartilage-like tissue. These results suggest fundamental differences between stem cell-based (MSC) and primary cell-based (hCH) tissue engineering, as well as the importance of suitable scaffold features, in the optimization of cartilage-related outcomes in vitro. The present work diversifies cell sources in combination with silk fibroin-based tissue engineering applications. Together with our previous studies, the present results show great promise for engineered 3-D silk fibroin scaffolds in autologous cell-based skeletal tissue engineering.

Animals↗

[Experimental study on constructing muscle tissue in rabbits with tissue engineering methods].

OBJECTIVE: To explore the possibility of constructing tissue engineering muscles by combining allogeneic myoblasts with small intestinal submucosa (SIS) in rabbits. METHODS: A large number of purified myoblasts were obtained with multi-procedure digestion and repeated attachment method from skeletal muscles taken from extremities of immature rabbits which were born 7 days ago. The myoblasts were labeled with BrdU, and then combined with SIS to construct tissue engineering muscles. This kind of tissue engineering muscles were grafted into the gastrocnemius muscle defect (1.5 cm in length, 1.0 cm in width) of fifteen rabbits as the experimental group. The SIS was grafted into the same position in the control group. The rabbits were sacrificed 4, 6, 8 weeks after operation. The tissue engineering muscles were evaluated by macroscopic, histological and immunohistochemical observations, and by quantitative analysis of local immunocyte in the grafting site. RESULTS: Allogeneic myoblasts with SIS were combined perfectly in vitro. The SIS was connected tightly to surrounding skeletal muscles and inflammation response was obvious 4 weeks after grafting. The SIS began to break down and inflammation response became slight 6 and 8 weeks after operation. Compared with that of 8th week, the quantitative analysis of local immunocyte in 4th and 6th week in both experimental and control group has significance (P<0.05). Newly formed muscle tissues were found around SIS in the experimental group in 4th, 6th, and 8th week. Expression of BrdU and myosin immunohistochemical staining were positive in the experimental group and negative in the control group. CONCLUSION: Tissue engineering muscles of rabbits which are constructed by combining allogeneic myoblasts with SIS can survive and proliferate.

Animals↗

[Fabrication of laryngeal cartilage by means of tissue engineering technique].

OBJECTIVE: To explore the method of fabricating tissue engineered laryngeal cartilage. METHODS: The rib and articular cartilage of infant New Zealand white rabbits were harvested in sterile condition. The chondrocytes were separated by collagenase digestion and cultured in vitro for 3 passage. Serial steps of solution casting, extrusion molding and particulate leaching were used to make larynx-shaped biomaterial models with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHH). The chondrocytes were seeded onto PHBHH scaffolds to form cell-PHBHH composites, which were subsequently in vitro for one week. After that, the measure of filling inner space of cell-PHBHH composites together with wrapping total composites using either greater omentum (n = 9) or fascia flap and muscle (also n = 9) in experimental groups was taken to implant the larynx-shaped biomaterial models seeded with chondrocytes into the belly and the back of adult New Zealand white rabbits. Control groups (every group n = 3) were the same measure as experimental groups but without chondrocyte on PHBHH scaffolds. Finally, morphological observation, HE staining & special staining and immunohistochemical test were conducted to assess cartilage regeneration and its shape at different period following implantation. RESULTS: The rate of viable cell in the final cell suspension was (93 +/- 2)% after well-controlled prolongation of digestion trypsin. Similar to that by traditional procedures (94 +/- 2)% (P > 0.05). The larynx-shaped PHBHH models with edges and corners of laryngeal cartilage made by us appeared to be hollow half-trumpet shape and its porosity was more than 90%. It showed that chondrocytes equally attached to the surface of porous PHBHH and filled within porousness with scanning electron microscopic examination. Tissue engineered larynx-shaped specimens could alternatively be harvested with the above mentioned two different implantation measures. The specimens presented to be similar to that before implantation in gross shape. It was demonstrated to be cartilaginous tissue through histological and immunohistochemical examination. Furthermore, There was nearly no difference between two kinds of tissue engineered laryngeal cartilage with two measures of implantation in morphology and histology. CONCLUSIONS: The regeneration of tissue engineered cartilage in vivo is not influenced by the chondrocytes harvested by improvement of well-controlled prolonged digestion with trypsin during in vitro cell culture. It seems that PHBHH may be used as scaffold in cartilage tissue engineering and wrapping together with filling method with either greater omentum or fascia flap and muscle is appropriate for fabricating tissue engineered laryngeal cartilage.

Animals↗

Vitrification as a prospect for cryopreservation of tissue-engineered constructs.

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.

Animals↗

Tissue engineering for periodontal regeneration.

As a result of periodontal regeneration research, a series of clinical techniques have emerged that permit tissue engineering to be performed for more efficient regeneration and repair of periodontal defects and improved implant site development. Historically, periodontal regeneration research has focused on a quest for "magic filler" material. This search has led to the development of techniques utilizing autologous bone and bone marrow, allografts, xenografts, and various man-made bone substitutes. Though these techniques have had limited success, the desire for a more effective regenerative approach has resulted in the development of tissue engineering techniques. Tissue engineering is a relatively new field of reconstructive biology which utilizes mechanical, cellular, or biologic mediators to facilitate reconstruction/regeneration of a particular tissue. In periodontology, the concept of tissue engineering had its beginnings with guided tissue regeneration, a mechanical approach utilizing nonresorbable membranes to obtain regeneration in defects. In dental implantology, guided bone regeneration membranes +/- mechanical support are used for bone augmentation of proposed implant placement sites. With the availability of partially purified protein mixture from developing teeth and growth factors from recombinant technology, a new era of tissue engineering whereby biologic mediators can be used for periodontal regeneration. The advantage of recombinant growth factors is this tissue engineering device is consistent in its regenerative capacity, and variations in regenerative response are due to individual healing response and/or poor surgical techniques. In this article, the authors review how tissue engineering has advanced and discuss its impact on the clinical management of both periodontal and osseous defects in preparation for implant placement. An understanding of these new tissue engineering techniques is essential for comprehending today's ever-expanding oral plastic surgery procedures.

Algorithms↗

Bioreactor design for tissue engineering.

Bioreactor systems play an important role in tissue engineering, as they enable reproducible and controlled changes in specific environmental factors. They can provide technical means to perform controlled studies aimed at understanding specific biological, chemical or physical effects. Furthermore, bioreactors allow for a safe and reproducible production of tissue constructs. For later clinical applications, the bioreactor system should be an advantageous method in terms of low contamination risk, ease of handling and scalability. To date the goals and expectations of bioreactor development have been fulfilled only to some extent, as bioreactor design in tissue engineering is very complex and still at an early stage of development. In this review we summarize important aspects for bioreactor design and provide an overview on existing concepts. The generation of three dimensional cartilage-carrier constructs is described to demonstrate how the properties of engineered tissues can be improved significantly by combining biological and engineering knowledge. In the future, a very intimate collaboration between engineers and biologists will lead to an increased fundamental understanding of complex issues that can have an impact on tissue formation in bioreactors.

Animals↗

Protein- and gene-based tissue engineering in bone repair.

A tissue engineering approach to bone regeneration includes the use of a scaffold, cells and bioactive factors alone or in various combinations. Several investigators have demonstrated enhanced bone formation when the tissue-engineered construct possesses traits inherent to autogenic bone grafts, namely osteoconductivity, osteoinductivity and osteogenicity. Use of the biodegradable polymer poly(lactide-co-glycolide) in combination with bone morphogenetic protein or primary cells genetically modified to release osteogenic protein have demonstrated the ability to induce osteogenic differentiation of, and subsequent mineralization by, muscle-derived cells and mesenchymal stem cells in both in vitro and in vivo applications.

Animals↗

Tissue-engineered autologous bladders for patients needing cystoplasty.

BACKGROUND: Patients with end-stage bladder disease can be treated with cystoplasty using gastrointestinal segments. The presence of such segments in the urinary tract has been associated with many complications. We explored an alternative approach using autologous engineered bladder tissues for reconstruction. METHODS: Seven patients with myelomeningocele, aged 4-19 years, with high-pressure or poorly compliant bladders, were identified as candidates for cystoplasty. A bladder biopsy was obtained from each patient. Urothelial and muscle cells were grown in culture, and seeded on a biodegradable bladder-shaped scaffold made of collagen, or a composite of collagen and polyglycolic acid. About 7 weeks after the biopsy, the autologous engineered bladder constructs were used for reconstruction and implanted either with or without an omental wrap. Serial urodynamics, cystograms, ultrasounds, bladder biopsies, and serum analyses were done. RESULTS: Follow-up range was 22-61 months (mean 46 months). Post-operatively, the mean bladder leak point pressure decrease at capacity, and the volume and compliance increase was greatest in the composite engineered bladders with an omental wrap (56%, 1.58-fold, and 2.79-fold, respectively). Bowel function returned promptly after surgery. No metabolic consequences were noted, urinary calculi did not form, mucus production was normal, and renal function was preserved. The engineered bladder biopsies showed an adequate structural architecture and phenotype. CONCLUSIONS: Engineered bladder tissues, created with autologous cells seeded on collagen-polyglycolic acid scaffolds, and wrapped in omentum after implantation, can be used in patients who need cystoplasty.

Adolescent↗

Monitoring local cell viability in engineered tissues: a fast, quantitative, and nondestructive approach.

Assessment of cell viability is a key issue in monitoring in vitro engineered tissue constructs. In this study we describe a fully automated, quantitative, and nondestructive approach, which is particularly suitable for tissue engineering. The approach offers several advantages above existing methods. Living and dead cell numbers can be separately determined for both isolated cells and cells that form networks during tissue formation. Moreover, viability can be locally monitored in time throughout the three-dimensional tissue. The viability assay is based on a dual fluorescent staining technique using CellTracker Green (CTG) for detection of living cells and propidium iodide (PI) for dead cells. CTG and PI images are created with a confocal laser scanning microscope. To determine the number of living cells, CTG fluorescence intensity is determined from the CTG image. Thereby, novel image-processing techniques have been developed, normalizing for various undesired influences that alter measurements of absolute CTG fluorescence intensities. Dead cell numbers are determined from the PI image, using an improved computerized counting method. The approach was first evaluated on C2C12 monolayers, of which images were taken directly after probe addition and 24 h later. Results show that at both times, computed living and dead cell numbers highly correlate with manually counted cell numbers (r > 0.996). Next, the approach was applied for monitoring viability in three-dimensional engineered skeletal muscle tissue constructs, which were subjected to unfavorable environmental conditions. This example illustrated that local viability can be quantitatively, nondestructively, and locally monitored in three-dimensional tissue constructs, making it a promising tool in the field of tissue engineering.

Animals↗

Tailored release of TGF-beta1 from porous scaffolds for cartilage tissue engineering.

In view of cartilage tissue engineering, the possibility to prepare porous scaffolds releasing transforming growth factor-beta(1) (TGF-beta(1)) in a well controlled fashion was investigated by means of an emulsion-coating method. Poly(ether-ester) multiblock copolymers were used to prepare emulsions containing TGF-beta(1) which were subsequently applied onto prefabricated scaffolds. This approach resulted in defined porous structures (66%) with interconnected porosity, suitable to allow tissue ingrowth. The scaffolds were effectively associated with TGF-beta(1) and allowed to tailor precisely the release of the growth factor from 12 days to more than 50 days by varying the copolymer composition of the coating. An incomplete release was measured by ELISA, possibly linked to the rapid concentration decrease of the protein in solution. The released growth factor retained its biological activity as was assessed by a cell proliferation assay and by the ability of the released protein to induce chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. However, exact bioactivity quantification was rendered difficult by the protein concentration decrease during storage. Therefore, this study confirms the interest of poly(ether-ester) multiblock copolymers for controlled release of growth factors, and indicates that emulsion-coated scaffolds are promising candidates for cartilage tissue engineering applications requiring precise TGF-beta(1) release rates.

Animals↗