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PubMed2003

Tissue engineering in urology: where are we going?

Tissue engineering in urology is a broad term used to describe the development of alternative tissue sources for diseased or dysfunctional native urologic tissue. This article reviews the recently published techniques involving synthetic and natural biodegradable matrices alone, known as "unseeded" scaffolds, and the latest data on "seeded" scaffolds, which are impregnated with cultured cells from urologic organs. Recent discoveries in reporter gene labeling of urologic tissue are discussed as a new method to identify and track the fates of these transplanted cells in vivo. This article also investigates how these bioengineering techniques are applied to synthetic and natural scaffolds, such as polyglycolic acid and porcine small intestine submucosa, to increase bladder capacity, repair urethral strictures, and replace corporal plaques in Peyronie's disease. Furthermore, recently published reports that these materials have been seeded with chondrocytes to create corporal rods for penile prostheses and stents for ureteral and urethral stricture disease are discussed. With these latest developments as a foundation, the future directions of tissue engineering in urology are presented.

Animals↗

PubMed2006

Rapid preparation of fresh frozen tissue-engineered bone sections for histological, histomorphological and histochemical analyses.

Fresh frozen sections are the best materials to assess tissue-engineered bone using cells/ceramic complexes. However, there are a lot of technical difficulties in obtaining serial sections suitable for microscopic examinations. Kawamoto et al. developed a method for the production of fresh frozen sections using new adhesive tape, and showed that sections were very useful for histological and histochemical studies. However, no study reported that the method was useful for tissue-engineered bone from histochemical and histomorphological points of view. This study aimed to determine the efficacy of fresh frozen sectioning for evaluating tissue-engineered bone. We revealed that fresh frozen sections retained the original morphology of tissue-engineered bone, and their biochemical characteristics. Therefore, rapid preparation of fresh frozen sections using adhesive tape is extremely useful for research of tissue-engineered bone, and serial sections can be assessed from both histomorphological and biochemical point of views. It is expected that this method will become a powerful tool in tissue-engineering of hard tissues.

Animals↗

PubMed2006

Monitoring of metabolite gradients in tissue-engineered constructs.

At present, the assessment of developing tissue-engineered constructs is almost always carried out destructively using biochemical or histological methods to determine cell number, viability and tissue growth throughout the construct. Since many of these experiments are long, taking weeks or even months to complete, simple and readily applicable non-destructive methods of monitoring changes in cell metabolism, viability and tissue deposition within the construct would be invaluable; such methods could point out adverse responses during the early stages of culture. Here, we describe the use of microdialysis for detecting local changes in cellular metabolism within a tissue-engineered construct. Three-dimensional constructs consisting of bovine articular chondrocytes entrapped in an alginate gel were cultured in a bioreactor for two weeks. Glucose and lactate were monitored by microdialysis, as the major nutrient and metabolite, respectively. Concentration gradients within the construct were evident, with the highest lactate concentrations in the construct centre. The local lactate concentration was a measure of cellular metabolic activity, decreasing as cellular activity fell and increasing as cellular activity was stimulated. Nutrient starvation and cell death in the construct centre could be readily detected in constructs deliberately cultured under adverse conditions. The results show that probe measurements can give an early warning of inappropriate local metabolic changes. Such information during the growth of tissue-engineered constructs would allow either corrective action or else an early end to an unsuccessful test.

Alginates↗

PubMed2005

[Repair of articular cartilage defects with "two-phase" tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells and "two-phase" allogeneic bone matrix gelatin].

OBJECTIVE: To investigate the effect of "two-phase" tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. METHODS: Thirty-two New Zealand white rabbits were involved in the experiment. "Two-phase" allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs were separated from 18 New Zealand white rabbits and induced to express chondrocytic phenotype. The chondrocyte precursor cells were seeded onto "two-phase" allogeneic BMG to construct tissue engineering cartilage. Masson's trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage (3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits (including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n = 18), with BMG at the left side (group B, n = 18), and without any implant at both sides in the remaining 9 rabbits as a control (group C, n = 18). After 1, 3 and 6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson's trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type I were carried out to assess the repair efficacy of tissue engineered cartilage. RESULTS: The "two-phase" BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 microm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 microm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage gradually thinned, but defect was repaired and the structure of the articular surface and subchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C (P < 0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type II in group A was also higher than those in groups B and C (P < 0.01). CONCLUSION: "Two-phase" allogeneic BMG is a prospective scaffold for tissue engineered cartilage, which combines with autologous chondrocyte precursor cells induced from MSCs to construct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.

Animals↗

PubMed2006

Tissue engineering cartilage with aged articular chondrocytes in vivo.

BACKGROUND: Tissue engineering has the potential to repair cartilage structures in middle-aged and elderly patients using their own "aged" cartilage tissue as a source of reparative chondrocytes. However, most studies on tissue-engineered cartilage have used chondrocytes from postfetal or very young donors. The authors hypothesized that articular chondrocytes isolated from old animals could produce neocartilage in vivo as well as articular chondrocytes from young donors. METHODS: Articular chondrocytes from 8-year-old sheep (old donors) and 3- to 6-month-old sheep (young donors) were isolated. Cells were mixed in fibrin gel polymer at 40 x 10 cells/ml until polymerization. Cell-polymer constructs were implanted into the subcutaneous tissue of nude mice and harvested at 7 and 12 weeks. RESULTS: Samples and native articular cartilage controls were examined histologically and assessed biochemically for total DNA, glycosaminoglycan, and hydroxyproline content. Histological analysis showed that samples made with chondrocytes from old donors accumulated basophilic extracellular matrix and sulfated glycosaminoglycans around the cells in a manner similar to that seen in samples made with chondrocytes from young donors at 7 and 12 weeks. Biochemical analysis revealed that DNA, glycosaminoglycan, and hydroxyproline content increased in chondrocytes from old donors over time in a pattern similar to that seen with chondrocytes from young donors. CONCLUSIONS: This study demonstrates that chondrocytes from old donors can be rejuvenated and can produce neocartilage just as chondrocytes from young donors do when encapsulated in fibrin gel polymer in vivo. This study suggests that middle-aged and elderly patients could benefit from cartilage tissue-engineering repair using their own "aged" articular cartilage as a source of reparative chondrocytes.

Age Factors↗

PubMed2001

Vascular tissue engineering.

The development of a tissue-engineered blood vessel substitute has motivated much of the research in the area of cardiovascular tissue engineering over the past 20 years. Several methodologies have emerged for constructing blood vessel replacements with biological functionality. These include cell-seeded collagen gels, cell-seeded biodegradable synthetic polymer scaffolds, cell self-assembly, and acellular techniques. This review details the most recent developments, with a focus on core technologies and construct development. Specific examples are discussed to illustrate both the benefits and shortcomings of each methodology, as well as to underline common themes. Finally, a brief perspective on challenges for the future is presented.

Animals↗

PubMed2003

[Expression of basic fibroblast growth factor and fibronectin in tissue engineering skin allograft during healing process].

OBJECTIVE: The aim of this study was to explore the expression of basic fibroblast growth factor (bFGF) and fibronectin during the healing process of allograft tissue engineering skin. METHODS: The tissue engineering skin that was obtained from neonatal SD rats was cultured in the lab. Afterwards, the skin was grafted into adult SD rats, and the expressions of bFGF and fibronectin were detected on the 7th, 10th, 14th, 20th and 30th day after the allograft of the tissue engineering skin. The autografted skin in 15 adult Wistar rats and the normal skin in 15 Wistar were treated as the control. HE staining and immunohistochemical staining were used to examine the healing of grafted skin. RESULTS: The expression of bFGF and fibronectin was the strongest on the 10th day after graft, and was weaker before the 10th day and after the 14th day. The expression changes of bFGF and fibronectin were similar as they were in the autograft group. CONCLUSION: The expression changes of bFGF and fibronectin in the tissue engineering skin during the process of wound healing were similar to those of autografted skin, and these can promote the repair of tissue engineering skin allograft.

Animals↗

PubMed2006

Cartilage tissue engineering: its potential and uses.

PURPOSE OF REVIEW: The prevalent nature of osteoarthritis, a cartilage degenerative disease that results in the erosion of joint surfaces and loss of mobility, underscores the importance of developing functional articular cartilage replacement. Recent research efforts have focused on tissue engineering as a promising approach for cartilage regeneration and repair. Tissue engineering is a multidisciplinary research area that incorporates both biological and engineering principles for the purpose of generating new, living tissues to replace the diseased/damaged tissue and restore tissue/organ function. This review surveys and highlights the current concepts and recent progress in cartilage tissue engineering, and discusses the challenges and potential of this rapidly advancing field of biomedical research. RECENT FINDINGS: Cartilage tissue engineering is critically dependent on selection of appropriate cells (differentiated or progenitor cells); fabrication and utilization of biocompatible and mechanically suitable scaffolds for cell delivery; stimulation with chondrogenically bioactive molecules introduced in the form of recombinant proteins or via gene transfer; and application of dynamic, mechanical loading regimens for conditioning of the engineered tissue constructs, including the design of specialized biomechanically active bioreactors. SUMMARY: Cell selection, scaffold design and biological stimulation remain the challenges of function tissue engineering. Successful regeneration or replacement of damaged or diseased cartilage will depend on future advances in our understanding of the biology of cartilage and stem cells and technological development in engineering.

Biocompatible Materials↗

PubMed2003

[Clinical application of tissue engineered bone repair of human craniomaxillofacial bone defects].

OBJECTIVE: To explore the feasibility of tissue engineered bone formation in human being using human bone marrow stromal cells (hBMSCs) and the possibility of clinical repair of craniomaxillofacial bone defects with tissue engineered bone. METHODS: Total 11 patients of cranial defects and aperture piriformis bone depression were included in this study. The hBMSCs were isolated by Percoll gradient centrifugation from patient's bone marrow aspirated from iliac crest. The hBMSCs were cultured in vitro and induced to become osteogenic cells in the DMEM medium containing 10% self-serum, beta-glycerophosphate (10 nmol/L) dexamethasone (10(-8) mol/L), L-2-ascorbic acid (50 micro mol/L), and 1, 25 (OH)(2)VD(3)(10 nmol/L). Induced hBMSCs of passage 3 were harvested and seeded onto partly demineralized allogenic bone matrix (pDBM) to form a cell-scaffold construct and in vitro co-culture for 1 week. The defects were repaired with the cell-scaffold construct. In 3 cases of aperture piriformis bone depression, one side was repaired with hBMSC/pDBM, while the other side was repaired by pDBM alone. All cases were followed up for 1, 3, 6 months post-operation as short-term evaluation and 1 to 2.5 years post-operation as long-term evaluation by three-dimensional computerized tomography (3D-CT) and clinical examination. In 2 cases who received secondary surgery, extra engineered bone tissue and control pDBM were harvested at the implantation sites for histological examination and immunohistochemistry. RESULTS: 3D-CT demonstrated that engineered bone was formed in 3 to 6 months post-operation. Additionally, formed bone maintained stable up to 1 - 2 years without absorption. Histologically, engineered bones revealed their structures similar to that of normal bone in HE staining. Interestingly, endochondral ossification was also observed in engineered bone. Immunohistochemistry shows positive staining of osteonectin and osteocalcin in engineered and normal bones. In contrast, implanted pDBM was completely degraded in 3 - 6 months as revealed by 3D-CT. Histologically, degraded pDBM and fibrous tissue were observed in the sites where pDBM alone was implanted. CONCLUSIONS: Tissue engineered bone can be formed in human being. Engineered bone can be used to repair clinical bone defect with satisfactory result. Furthermore, the result of this study proves that tissue engineered bone is possible for clinical application.

Adolescent↗

PubMedDate not supplied

Synergy between genetic and tissue engineering: Runx2 overexpression and in vitro construct development enhance in vivo mineralization.

Tissue engineering has emerged as a promising strategy to generate bone-grafting substrates. These approaches, however, are limited by an insufficient supply of committed osteoprogenitor cells and dedifferentiation of osteogenic cells during in vitro culture. To address these limitations, we engineered bone marrow stromal cells to constitutively express the osteoblastic transcription factor Runx2/Cbfa1, using retroviral gene delivery. These Runx2-modified cells were integrated into three-dimensional polymeric scaffolds to create tissue-engineered constructs. Compared with control stromal cells, Runx2 overexpression significantly upregulated osteoblastic differentiation and mineralization in vitro and in vivo in an ectopic, nonosseous subcutaneous site. More importantly, in vitro construct development to create a mineralized template before implantation dramatically enhanced subsequent in vivo mineralized tissue formation, providing a novel templating tissue-engineering strategy to improve in vivo mineralization. Finally, Runx2 overexpression and in vitro construct development synergistically enhanced in vivo mineralization compared with in vitro construct development or genetic engineering alone. This work provides a novel integrated genetic and tissue-engineering strategy to create mineralized templates for generating robust bone-grafting material.

Animals↗

PubMed2006

Biopolymer-based biomaterials as scaffolds for tissue engineering.

Biopolymers as biomaterials and matrices in tissue engineering offer important options in control of structure, morphology and chemistry as reasonable substitutes or mimics of extracellular matrix systems. These features also provide for control of material functions such as mechanical properties in gel, fiber and porous scaffold formats. The inherent biodegradability of biopolymers is important to help regulate the rate and extent of cell and tissue remodeling in vitro or in vivo. The ability to genetically redesign these polymer systems to bioengineer appropriate features to regulate cell responses and interactions is another important feature that offers both fundamental insight into chemistry-structure-function relationships as well as direct utility as biomaterials. Biopolymer matrices for biomaterials and tissue engineering can directly influence the functional attributes of tissues formed on these materials and suggest they will continue play an increasingly important role in the field.

Animals↗

PubMed2004

Functional innervation in tissue engineered models for in vitro study and testing purposes.

The biotechnology industry is rapidly expanding and the emerging field of tissue engineering is projected to have a high impact in the near future. Recently the field of cellular, drug, and prosthetic delivery has melded with the field of tissue engineering to make simulated tissues. In addition to their roles as tissue substitutes for transplantation, these simulated tissues may provide more accurate models and environments for toxicology testing and the study of peripheral nerves. The current study demonstrates the importance of innervation, in general, for the function of engineered tissues. We observe that the presence of nerves in a tissue engineered (TE) human cornea model enhances the growth of the epithelium and the formation of its protective mucin layer. Innervation also confers protection to the epithelium from chemical insult, as determined by the level of post-treatment epithelial cell death. We demonstrate differential responses of the nerves to chemical stimuli by changes in intracellular sodium as measured by 2-photon microscopy. The 2-photon imaging techniques also allow for the visualization and study of the fine sensory axon fibers within the 3-dimensional tissue. This work demonstrates a role for innervation in the protective quality and function of the engineered tissue, and the potential to use the nerves themselves as indicators of the severity of an insult. These results are important to consider for the development of any optimized TE models for in vitro study and testing purposes.

Animals↗

PubMed2005

Chitosan-alginate hybrid scaffolds for bone tissue engineering.

A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Here we report on the development of a biodegradable porous scaffold made from naturally derived chitosan and alginate polymers with significantly improved mechanical and biological properties as compared to its chitosan counterpart. Enhanced mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan-alginate scaffold, proliferated well, and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The chitosan-alginate scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation. Coacervation of chitosan and alginate combined with liquid-solid separation provides a scaffold with high porosity, and mechanical and biological properties suitable for rapid advancement into clinical trials.

Alginates↗

PubMed1999

Emerging approaches to the tissue engineering of fat.

The eventual development of tissue-engineered fat equivalents for reconstructive and augmentation purposes will be most welcome by nearly every surgical discipline and prove to be especially useful for plastic surgeons. The clinical applications for which tissue-engineered fat will be particularly useful are vast and varied and can be loosely categorized into reconstructive, cosmetic, corrective, and orthotic indications. In this article, the authors discuss the emerging tissue-engineering strategies for fat, including the procurement of autologous cells, cell growth and differentiation, implantation and engraftment, polymer scaffolds, and implant integration and histogenesis.

Adipocytes↗

PubMed1999

Ligament tissue engineering using synthetic biodegradable fiber scaffolds.

Tissue engineering offers the possibility of replacing damaged human ligaments with engineered ligament tissues. Hence, we attempted to culture in vitro ligament tissues by seeding human anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells onto synthetic biodegradable polymer fiber scaffolds. The ACL and MCL cells readily attached to the scaffold fibers. These cells and their secreted matrix soon surrounded the scaffold fibers and bridged the gaps in between. Beginning at 2 weeks, portions of the scaffolds were completely filled with tissue matrix. By 5 weeks, the scaffolds became single bundles of tissue. Thus the cell/fiber system appears to be a viable system for culturing ligament tissues. Additionally, cell proliferation under mechanical and biochemical stimuli was studied for up to 4 days. Whereas mechanical stimulus and transforming growth factor enhanced proliferation, inflammatory agents (lipopolysaccharide and complement C5a) had a negative effect. This work can thus contribute to a sound strategy for culturing replacement ligament tissues in vitro.

Anterior Cruciate Ligament↗

PubMed2005

A method for solvent-free fabrication of porous polymer using solid-state foaming and ultrasound for tissue engineering applications.

Most of the existing fabrication techniques for tissue engineering scaffolds require the use of organic solvents that may never be fully removed even after long leaching hours. The residues of these organic solvents reduce the ability of biological cells to form new tissue. This paper presents an approach toward solvent-free fabrication of tissue engineering scaffolds. Interconnected porous structures were created using solid-state foaming and ultrasound. The material used in this study was polylactic acid (PLA) and the blowing agent was CO(2). In order to determine suitable process conditions, saturation and foaming studies were first conducted. Selected foam samples were then processed using pulsed ultrasound. The microstructures before and after the ultrasound processing were compared. It was shown that the inter-pore connectivity of the solid-state foams was substantially enhanced. The combined solid-state foaming and ultrasound processing provide a way to fabricate porous polymer for potential tissue engineering applications.

Biocompatible Materials↗

PubMed2005

The tissue-engineered vascular graft using bone marrow without culture.

OBJECTIVE: To overcome the shortcomings of current vascular grafts, tissue-engineering methods have been applied to cardiovascular regions. We previously reported the creation of a tissue-engineered vascular graft by using vascular mixed cells. However, the cost and manpower for harvesting and culturing the cells was too burdensome. To overcome these drawbacks, we have developed a new method for creating a tissue-engineered vascular graft by using bone marrow cells, which can be obtained easily and used immediately, without cell culture. METHODS: Biodegradable polymers seeded with different types of cells (group V, cultured venous cells; group B, bone marrow cells without culture; and group C, non-cell-seeded graft [as control]) were implanted into the inferior venae cavae of dogs. The grafts were explanted at 4 weeks and assessed histologically and biochemically. RESULTS: In the histologic examination, a regular layer of Masson-staining collagen fiber and a layer of factor VII-stained endothelial and ant-alpha-smooth muscle cell antigen-immunoreactive cells stained in groups V and B like native vascular tissue, whereas no such stained regular lining was detected in group C. A 4-hydroxyproline assay in group C showed significantly lower levels than in groups V and B or native tissue ( P < .05). The DNA content of the tissue-engineered vascular graft tended to be higher in group C than in groups V and B or in native tissue. CONCLUSIONS: In the creation of tissue-engineered vascular grafts, the method of using bone marrow cells seems to be useful and superior to that of using vascular cells because bone marrow cells can be used directly, without culture.

Animals↗

PubMed1998

European research and commercialisation activities in the field of tissue engineering and liver support in world wide competition.

Tissue engineering is seen as an interesting field of technology which could improve medical therapy and could also be considered as a commercial opportunity for the European biotechnological industry. Research in the state of the art of science using the MedLine and the Science Citation Index databases, in the patent situation and of the industry dealing with tissue engineering was done. A special method, based on the Science Citation Index Journal Citation Report 1993, for evaluating scientific work was defined. The main countries working in the field of tissue engineering were evaluated in regard to their scientific performance and their patents. The R&D of German industry was investigated as an exemplary European country. Out of all activities, different tissues were rated with respect to the attention received from research and industry and with regard to the frequency in which patents were applied for. USA, Germany and Japan rank first in most tissues, especially liver. After comparing German patents with the German scientific and industrial work, it seems that the potential in German patents and research is underestimated by German industry and inefficiently exploited.

Artificial Organs↗