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Tissue engineering of stratified articular cartilage from chondrocyte subpopulations.

OBJECTIVE: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. ESIGN: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). RESULTS: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. CONCLUSIONS: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage.

Animals↗

Cell-interactive alginate hydrogels for bone tissue engineering.

There is significant interest in the development of injectable carriers for cell transplantation to engineer bony tissues. In this study, we hypothesized that adhesion ligands covalently coupled to hydrogel carriers would allow one to control pre-osteoblast cell attachment, proliferation, and differentiation. Modification of alginate with an RGD-containing peptide promoted osteoblast adhesion and spreading, whereas minimal cell adhesion was observed on unmodified hydrogels. Raising the adhesion ligand density increased osteoblast proliferation, and a minimum ligand density (1.5-15 femtomoles/cm2) was needed to elicit this effect. MC3T3-E1 cells demonstrated increased osteoblast differentiation with the peptide-modified hydrogels, as confirmed by the up-regulation of bone-specific differentiation markers. Further, transplantation of primary rat calvarial osteoblasts revealed statistically significant increases of in vivo bone formation at 16 and 24 weeks with G4RGDY-modified alginate compared with unmodified alginate. These findings demonstrate that biomaterials may be designed to control bone development from transplanted cells.

3T3 Cells↗

Cutaneous tissue engineering and lower extremity wounds (part 1).

Tissue-engineered skin is a novel therapeutic with which difficult-to-heal lower extremity wounds may be treated. Such skins are products of cutaneous tissue engineering that provide an alternative for autologous or allogeneic tissue transplantation, thereby avoiding problems associated with donor site availability, the risk of infection, and scarring. Recently developed tissue-engineered skin equivalents have shown to be superior in certain ways to compression therapy for refractory venous ulcers and acute wounds. These biologic products behave similarly to autografts.

Journal Article↗

[Tissue-engineered cartilage in a prefabricated microvascularized flap].

INTRODUCTION: 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 a particular defect. The aim of the present study was to establish the technique of prefabricating a microvascular free flap by implanting a vessel loop under a skin flap in a rabbit model. The second aim was to gather experience with prelaminating the flap with autologous tissue-engineered cartilage in terms of matrix development, inflammatory reaction and host-tissue interaction. METHODS: The microvascular flap was created 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 2-3 weeks. One cell-biomaterial construct was placed beneath the prefabricated flap, and the others were placed subcutaneously under the abdominal skin and intermuscularly at the lower extremity. In addition, a biomaterial sample without cells was placed subcutaneously as a control. All implanted specimens were left in position for 6 or 12 weeks. After explantation, the specimens were examined by histological and immunohistological methods. The prefabricated flap was analyzed by angiography. RESULTS: The prefabricated flaps showed a well-developed network of blood vessels formed by neovascularization 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 hints of inflammatory reactions were observed. CONCLUSION: These results show the potential of prefabricated flaps as custom-made flaps for reconstructive surgery in difficult circumstances, more or less independent of anatomical prerequisites. Cartilage tissue engineering provides a 3-dimensional structure with minimal donor-site morbidity.

Animals↗

Tissue engineering using adult stem cells.

Patients with a variety of diseases may be treated with transplanted tissues and organs. However, there is a shortage of donor tissues and organs, which is worsening yearly because of the aging population. Scientists in the field of tissue engineering are applying the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. The stem cell field is also advancing rapidly, opening new options for cellular therapy and tissue engineering. The use of adult stem cells for tissue engineering applications is promising. This chapter discusses applications of these new technologies for the engineering of tissues and organs. The first part provides an overview of regenerative medicine and tissue engineering techniques; the second highlights different adult stem cell populations used for tissue regeneration.

Adult Stem Cells↗

A new source for cardiovascular tissue engineering: human bone marrow stromal cells.

OBJECTIVE: Vascular-derived cells represent an established cell source for tissue engineering of cardiovascular constructs. Previously, cell isolation was performed by harvesting of vascular structures prior to scaffold seeding. Marrow stromal cells (MSC) demonstrate the ability to differentiate into multiple mesenchymal cell lineages and would offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. We studied the feasibility of using MSC as an alternative cell source for cardiovascular tissue engineering. METHODS: Human MSC were isolated from bone marrow and expanded in culture. Subsequently MSC were seeded on bioabsorbable polymers and grown in vitro. Cultivated cells and seeded polymers were studied for cell characterization and tissue formation including extracellular matrix production. Applied methods comprised flow cytometry, histology, immunohistochemistry, transmission (TEM) and scanning electron microscopy (SEM), and biochemical assays. RESULTS: Isolated MSC demonstrated fibroblast-like morphology. Phenotype analysis revealed positive signals for alpha-smooth muscle actin and vimentin. Histology and SEM of seeded polymers showed layered tissue formation. TEM demonstrated formation of extracellular matrix with deposition of collagen fibrils. Matrix protein analysis showed production of collagen I and III. In comparison to vascular-derived cell constructs quantitative analysis demonstrated comparable amounts of extracellular matrix proteins in the tissue engineered constructs. CONCLUSIONS: Isolated MSC demonstrated myofibroblast-like characteristics. Tissue formation on bioabsorbable scaffolds was feasible with extracellular matrix production comparable to vascular-cell derived tissue engineered constructs. It appears that MSC represent a promising cell source for cardiovascular tissue engineering.

Actins↗

[Therapeutic effect of tissue engineered tendon in repairing old calcaneal tendon rupture and defects].

OBJECTIVE: To probe into the surgical methods and therapeutic effect of repairing old calcaneal tendon rupture and defects with tissue engineered tendons. METHODS: The tissue engineered tendons were prefabricated by co-cultivating allogeneic tendon cells with composite of carbon fiber and polyglycolic acid for 5 days. From August 1999 to June 2002, 7 patients with calcaneal tendon rupture and defects (5-7 cm in length) were treated with tissue engineered tendons. The defects were repaired by suturing repeatedly with tissue engineered tendons. Meanwhile, the defects were covered by gastrocnemius fascial flap for protection and strengthening. After surgery, the ankle joints were fixed with plaster 4 to 6 weeks, and then the functional exercise was done. RESULTS: All the patients were followed up 22 to 56 months (46.9 months on average). Six patients achieved healing by the first intention, only one patient had delayed union. No local or systemic complication occurred in all the cases. No patients were given the second operation for adhesion. In accordance with YIN Qingshui's criterion for therapeutic effect, the results were excellent in 5 cases, good in 1 case and moderate in 1 case. CONCLUSION: Repairing old rupture and defects of calcaneal tendon with tissue engineered tendons can achieve good clinical outcome, it is an optional therapy.

Achilles Tendon↗

Growth factor release from tissue engineering scaffolds.

Synthetic scaffold materials are used in tissue engineering for a variety of applications, including physical supports for the creation of functional tissues, protective gels to aid in wound healing and to encapsulate cells for localized hormone-delivery therapies. In order to encourage successful tissue growth, these scaffold materials must incorporate vital growth factors that are released to control their development. A major challenge lies in the requirement for these growth factor delivery mechanisms to mimic the in-vivo release profiles of factors produced during natural tissue morphogenesis or repair. This review highlights some of the major strategies for creating scaffold constructs reported thus far, along with the approaches taken to incorporate growth factors within the materials and the benefits of combining tissue engineering and drug delivery expertise.

Bioartificial Organs↗

Decision support for tendon tissue engineering.

The aim of this study is to provide decision support with artificial intelligence for tendon tissue engineering strategies. The experimental data of tissue-engineered tendons were integrated and standardized with a centralized database, and a decision support system was developed using both artificial neural networks and decision trees. The decision support system was trained with existing cases in the database, and then was used to generate tissue engineering schemes for new experimental animals. Following the schemes generated by the artificial intelligent system, we cured 28 of the 30 experimental animals. In conclusion, artificial intelligence is a powerful method for decision support in the tendon tissue engineering realm.

Animals↗

Mesenchymal stem cells in musculoskeletal tissue engineering: a review of recent advances in National University of Singapore.

A key factor in the tissue engineering approach to tissue repair and regeneration is the use of appropriate cells. Mesenchymal stem cells (MSCs) are derived from bone marrow stroma or connective tissues and they have the potential to differentiate into various mesenchymal cell lines in vitro and in vivo. These cells hold great promise for musculoskeletal tissue engineering. This review is based mainly on the work which has been done in the National University of Singapore on the use of MSCs for engineering cartilage, growth plate, bone and tendon/ligament as well as the clinical trail of autologous chondrocyte implantation. It can help to shape future research on musculoskeletal tissue engineering.

Animals↗

Biocompatibility of poly-DL-lactic acid (PDLLA) for lung tissue engineering.

This study explores the possibility of growing lung cells on poly-DL-lactic acid (PDLLA) scaffolds, with a view to in future engineer pulmonary tissue for human implantation. As a first step in this process, the ability of PDLLA to maintain the growth of lung epithelium is tested using a robust cell line. Poly-DL-lactic acid has been investigated in two forms, as planar discs and as 3-D foams, and it has been demonstrated that PDLLA is not only nontoxic to pneumocytes but it also actively supports their growth. The initial findings suggest that the material is an appropriate matrix for engineering of distal lung tissue.

Animals↗

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

Animals↗

Surface adsorption of DNA to tissue engineering scaffolds for efficient gene delivery.

Gene delivery from tissue engineering scaffolds has potential to promote localized transgene expression that can induce the formation of functional tissues. Substrate-mediated delivery, an alternative delivery strategy to sustained release, is based on immobilization of DNA complexes to the polymer surface for subsequent delivery to cells cultured on the substrate. We investigate polyethylenimine (PEI)/DNA complex immobilization and subsequent cellular transfection on tissue engineering scaffolds fabricated from poly(lactide-co-glycolide) (PLG). The properties of the substrate and the complex affect both immobilization and cellular transfection. PLG promotes binding of PEI/DNA complexes, with percent bound independent of the N/P ratio or the DNA dosage. The levels of transgene expression are similar to or greater than control studies based on bolus DNA delivery, with orders of magnitude less DNA. Immobilization also homogeneously distributes the DNA throughout the scaffold, resulting in large numbers of transfected cells (>60%) at low surface quantities (<50 ng). Importantly, this approach can be employed to transfect cells throughout a three-dimensional scaffold. Tissue engineering scaffolds that are prefabricated into various shapes from a range of materials could potentially employ this strategy for numerous applications.

Adsorption↗

[Tissue engineering of skin].

OBJECTIVE: To review the recent progresses on tissue engineering of skin. METHODS: Recent original articles about tissue engineering of skin were extensively reviewed, which focused on the progresses and major problems concerning the epidermal substitutes, dermal substitutes, cultured-epidermal composite skin graft. RESULTS: Most investigators had come to conclusion that the optimal skin substitute should provide for immediate reconstruction of both the lost epidermis and dermis. The research was mainly focused on how to transplant epidermal cells immediately, preserve their activity and function, and develop the extracellular matrix which could effectively accelerate the function of transplanted cells, induce vascular growth from the wound bed, could be biodegradable, no toxicity and no danger of carrying pathogen. CONCLUSION: The major research trends of tissue engineering of skin should be focused on the study of immediate transplantation of epidermal cells, accelerate wound healing and developing extracellular matrix of dermis.

Cell Transplantation↗

Myocardial tissue engineering: creating a muscle patch for a wounded heart.

Cardiac tissue engineering promises to revolutionize the treatment of patients with end-stage heart failure and provide new solutions to the serious problem of heart donor shortage. By its broad definition, tissue engineering involves the construction of tissue equivalents from donor cells seeded within three-dimensional polymeric scaffolds, then culturing and implanting of the cell-seeded scaffolds to induce and direct the growth of new, healthy tissue. Here, we present an up-to-date summary of research studies in cardiac tissue engineering, with an emphasis on the critical design principles.

Cardiomyopathies↗

Implantation of preadipocyte-loaded hyaluronic acid-based scaffolds into nude mice to evaluate potential for soft tissue engineering.

The reconstruction of soft tissue defects following extensive deep burns or tumor resections remains an unresolved problem in plastic and reconstructive surgery since adequate implant materials are still not available. Preadipocytes, immature precursor cells found between mature adipocytes in adipose tissue, are a potential material for soft tissue engineering since they can proliferate and differentiate into adipose tissue after transplantation. In previous studies, we identified hyaluronan benzyl ester (HYAFF 11) sponges to be promising carrier matrices. This study now evaluates, in vitro and in vivo, a new sponge architecture with pores of 400 microm either made of plain HYAFF 11 or HYAFF 11 coated with the extracellular matrix glycosaminoglycan hyaluronic acid. Human preadipocytes were isolated, seeded onto carriers and implanted into nude athymic mice. Explants harvested after 3, 8, and 12 weeks were examined for macroscopical appearance, thickness, weight, pore structure, histology, and immunohistochemistry. Compared to previous studies, we found better penetration of cells into both types of scaffolds, with more extensive formation of new vessels throughout the construct but with only minor adipose tissue. Our encouraging results contribute towards a better seeded and vascularised scaffold but also show that the enhancement of adipogenic conversion of preadipocytes remains a major task for further in vivo experiments.

Adipocytes↗

Effect of a degraded core on the mechanical behaviour of tissue-engineered cartilage constructs: a poro-elastic finite element analysis.

The structure and functionality of tissue-engineered cartilage is determined by the tissue culture conditions and mechanical conditioning during growth. The quality of tissue-engineered cartilage can be evaluated using tests such as the confined compression test. Tissue-engineered cartilage constructs usually consist of an outer layer of cartilage and an inner core of either undeveloped cartilage or degrading scaffold material. A biphasic poro-elastic finite element model was used to demonstrate how such a core influences the reaction force-time curve obtained from a confined compression test. The finite element model predicted that higher volumes of degraded scaffold in the inner core would reduce the aggregate modulus calculated from the confined compression test and raised the estimate of tissue permeability. The predicted aggregate modulus reduced from 0.135 MPa, for a homogenous construct, to 0.068 MPa, for a construct that was only 70% cartilaginous. It was found that biphasic poro-elastic finite modelling should be used in preference to a one-dimensional model that assumed homogeneity in estimating the properties of tissue-engineered cartilage.

Biomechanical Phenomena↗