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PubMed2005

Effect of donor characteristics, technique of harvesting and in vitro processing on culturing of human marrow stroma cells for tissue engineered growth of bone.

The aim of this study was to assess the effect of donor characteristics and the technique of harvesting and in vitro processing on the efficacy of culturing of human mesenchymal stem cells (hMSCs) for tissue engineered growth of bone. Cultures of hMSCs were derived from iliac crest bone marrow aspirates (21 donors, age 11-76) and from cancellous bone grafting material (32 donors, age 13-84). Age had no significant effect on the ability to isolate and culture hMSCs, although the failure rate was 55.6% in donors beyond the age of 60, while it varied between 14.3% and 22.2% in donors under 60 years of age. Male and female donors had comparable failure rates (27.3% and 28.6%, respectively). Culturing of hMSCs was successful in 90.4% of marrow aspirates from 21 donors and in 62.5% of cancellous bone specimens from 35 donors. This difference was statistically significant (P=0.023). Regression analysis confirmed that at simultaneous testing of the three variables, only the source of cells significantly affected the result (P=0.043). Morphological evaluation of the unfractionated primary population showed a change in cell shape of the adherent cells from a triangular into thin spindle-shaped elongated form, which remains until confluence. When the cultures were exposed to osteoinductive medium, various morphotypes expressing different levels of alkaline phosphatase and secreting different amounts of mineral were evident. Morphology of marrow stroma cells (MSCs) from marrow aspirates was not different from MSCs derived from cancellous bone specimens. Expression of osteogenic markers in MSCs as shown by PCR as well, did not differ between the two sources. It is concluded that marrow aspirates and cancellous bone specimens produce comparable populations of MSCs. However, bone marrow aspirates from donors under the age of 60 years rather than cancellous bone chips are favourable for isolation and expansion of hMSCs for tissue engineered growth of bone.

Adolescent↗

PubMed2006

Fluid flow increases type II collagen deposition and tensile mechanical properties in bioreactor-grown tissue-engineered cartilage.

A novel parallel-plate bioreactor has been designed to apply a consistent level of fluid flow-induced shear stress to tissue-engineered articular cartilage in order to improve the matrix composition and mechanical properties and more nearly approximate to that of native tissue. Primary bovine articular chondrocytes were seeded into the bioreactor at high densities (1.7 x 10(6) cell/cm2) without a scaffold and cultured for two weeks under static, no-flow conditions. A mean fluid flow-induced shear stress of 1 dyne/cm2 was then applied continuously for 3 days. The application of flow produced constructs with significantly (p < 0.05) higher amounts of total collagen (via hydroxyproline) and specifically type II collagen (via ELISA) (25.3 +/- 2.5% and 22.1 +/- 4.7% of native tissue, respectively) compared to static controls (22.4 +/- 1.7% and 9.5 +/- 2.3%, respectively). Concurrently, the tensile Young's modulus and ultimate strength were significantly increased in flow samples (2.28 +/- 0.19 MPa and 0.81 +/- 0.07 MPa, respectively) compared to static controls (1.55 +/- 0.10 MPa and 0.62 +/- 0.05 MPa, respectively). This study suggests that flow-induced shear stresses and/or enhanced mass transport associated with the hydrodynamic environment of our novel bioreactor may be an effective functional tissue-engineering strategy for improving matrix composition and mechanical properties in vitro.

Animals↗

PubMedDate not supplied

Symposium on Metrology and Standards for Cell Signaling: Impact on Tissue Engineering, National Institute for Standards and Technology, October 14-15, 2003.

The Symposium on Metrology and Standards for Cell Signaling: Impact on Tissue Engineering, was cosponsored by the National Institute for Standards and Technology (NIST) and ASTM International, and was held at the NIST on October 14 and 15, 2003. The purpose of the symposium was to discuss the role of standards and quantitative measurements of cellular responses for the field of tissue engineering. Accurate and quantitative measurements, reference materials, and standards are needed to facilitate the understanding and control of cell responses to materials, culture media, and the in vivo environment. Speakers included government researchers from the NIST, the Food and Drug Administration, and the National Cancer Institute; vice presidents, chief executive officers, and directors of research from companies; and academic researchers in engineering, medicine, and cell biology. The metrology needs identified include quantitative assays of cell signaling pathways, bioinformatics that will allow consolidation of these complex data and permit correlative parameters to be identified, well-characterized standardized cell lines against which responses in other cells can be compared, and reference materials for use in control experiments.

Cell Culture Techniques↗

PubMed2005

New perspectives of penile enhancement surgery: tissue engineering with biodegradable scaffolds.

OBJECTIVE: To evaluate in a multicenter, prospective study preliminary aesthetic and functional results of autologous ex-vivo tissue engineering for penile girth enhancement. METHODS: From July 1999 to January 2004, 204 men of mean age 26.77 (range 19-54 years) underwent this procedure. Indications for penile girth enhancement were penile dysmorphic disorder and previous failed surgery for penile girth enhancement. Fibroblast cells harvested from 1 cc of biopsied scrotal dermal tissue were expanded in culture until the total cell number of at least 2x10(7) was reached. Suspended cells in culture medium were then seeded on pretreated tube-shaped PLGA scaffolds and incubated for 24 hours. After penile degloving, scaffolds were shape adjusted and transplanted between dartos and Buck's fascia when the skin was compliant or under the neurovascular bundle when the skin was not compliant. RESULTS: A total of 84 randomly selected patients were followed 1 to 5 years postoperatively (median 24 months). The gain in girth ranged from 1.9 to 4.1cm (mean 3.15 cm). Postoperative complications occurred as infection in three, penile skin pressure necrosis in two and seroma formation in five patients and were all treated conservatively. Surgical intervention was appraised by patients on a scale from 1 to 5 as follows: the best mark (5) was given by 44.05%, very good (4) by 36.90%, good (3) by 19.05% and only one patient gave the mark 2 judging general penile appearance as dissatisfactory; mean score was 4.25. CONCLUSION: Autologous tissue engineering by using biodegradable scaffolds as a carrier is a new and safe therapeutic approach for penile girth enhancement. The outcome of this study points out the necessity for its expanded clinical applicability in the future.

Absorbable Implants↗

PubMed2005

Performance of polyoxymethylene plastic (POM) as a component of a tissue engineering bioreactor.

Polyoxymethylene (POM, acetal homopolymer, polyacetal), commercialized as Delrin by DuPont, is an engineering resin with mechanical properties that make it useful for the prototyping and manufacture of laboratory apparatus. These properties include excellent, "metal-like," machining characteristics and dimensional stability, as well as thermal stability, which allows steam sterilization. Historically, POM has been used widely, including as a surgical implant material. For these reasons, we have used this plastic as a media-wetted component in a tissue-engineering bioreactor, with good results. However, a study by LaIuppa et al.5 suggested that POM is unsuitable for use in a cell culture environment (LaIuppa et al. J Biomed Mater Res 1997;36:347-359). POM is based on the polymerization of formaldehyde, and, in addition, contains stabilizers and/or fillers. All of these could potentially be released into the medium, e.g., as formaldehyde or other thermal breakdown products, especially upon repeated autoclaving. The cited report thus appeared plausible, although contrary to our observations. In this study, we specifically assessed whether media conditioned by long-term exposure to machined white POM had a negative effect on the proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). We selected this cell system, as cartilage tissue engineering is the primary application of our bioreactor system. The POM samples were steam-autoclaved 1 to 20 times, to assess the possibility of any toxic thermal breakdown product release into the media. We found that MSCs did not attach directly to machined POM. Because cells that escape from the tissue construct cannot colonize the reactor and compete for nutrients, this is a desirable characteristic of a material used in a tissue-engineering bioreactor. Furthermore, the use of POM-conditioned media had no detectable impact on the proliferation rate of MSCs measured over a one-week period; nor was any effect on chondrogenic differentiation observed at up to 3 weeks in culture. In summary, the use of POM as a culture medium-wetted component appears to be innocuous, at least for human MSCs. The contrast of these findings to those of LaIuppa et al.5 may reflect a cell-type specific sensitivity, or may be due to different handling of the material.

Acetals↗

PubMedDate not supplied

Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments.

Bone marrow stromal cells (BMSCs) play a central role in the repair and regeneration of mesenchymal tissues. For tissue engineering of ligaments and tendons, both stimulation of cell proliferation and differentiation with increased expression of essential extracellular matrix proteins and cytoskeletal elements are desirable. This study analyzes the effect of low-dose (3 ng/mL) fibroblast growth factor 2 (FGF-2) and high-dose FGF-2 (30 ng/mL) on proliferation (bromodeoxyuridine content, spectrophotometry), differentiation (transcription of collagen I, collagen III, fibronectin, elastin, alpha-smooth muscle actin, and vimentin, reverse transcription-polymerase chain reaction, and cell density and apoptosis (annexin V, fluorescence-activated cell sorting) of human BMSCs, and compares the results with those of a control group without FGF-2. Low-dose FGF-2 triggered a biphasic BMSC response: on day 7, cell proliferation reached its maximum and was significantly higher compared with the other groups. On days 14 or 28, collagen I, collagen III, fibronectin, and alpha- smooth muscle actin mRNA expression was significantly enhanced in the presence of low-dose FGF-2. In contrast, high-dose FGF-2 did not stimulate differentiation or proliferation. Vimentin mRNA was expressed only in cultures with low-dose and high-dose FGF-2 after 14 and 28 days. Cell density was significantly higher in cultures with low-dose FGF-2 compared with the group with high-dose FGF-2 on days 7, 14, and 28. The apoptosis rate remained stable, at a rather high level, in all groups. Microscopic investigation of the cell cultures with low-dose FGF-2 showed more homogeneous, dense, fibroblast-like, spindle-shaped cells with long cell processes compared with cultures with high-dose, or no FGF-2. Low-dose FGF-2 may be useful for tissue engineering of ligaments and tendons by increasing BMSC proliferation and stimulating mRNA expression of specific extracellular matrix proteins and cytoskeletal elements.

Bone Marrow Cells↗

PubMedDate not supplied

Initial assessment of a tissue engineered stomach derived from syngeneic donors in a rat model.

The objective of this study is to assess the feasibility of creating a tissue engineered stomach using isolated stomach epithelium organoid unit from syngeneic adult donors and a biodegradable polymer scaffold in a rat model. Despite recent advances in reconstruction techniques, total gastrectomy is still accompanied by various complications. As an alternative treatment, a tissue engineered stomach that replaces the mechanical and metabolic functions of a normal stomach is proposed. Stomach epithelium organoid units were isolated from syngeneic adult rats and seeded onto biodegradable polymers. These constructs were implanted into the omenta of recipient adult rats. All constructs were harvested for histologic and immunohistochemical examination at designated time points. Cyst-like structures were formed that showed the development of vascularized tissue with a neomucosa. Immunohistochemical staining for alpha-actin smooth muscle, gastric mucin, and proton pump indicated the presence of a smooth muscle layer and gastric epithelium, as well as the existence of parietal cells of the stomach mucosa, respectively. Epithelium derived stomach organoid units seeded on biodegradable polymers were transplanted in donor rats and have been shown to vascularize, survive, and regenerate into complex tissue resembling a native stomach. These initial results are encouraging, and studies are currently underway to further assess this approach.

Absorbable Implants↗

PubMed2000

Tissue engineering from Adam to the zygote: historical reflections.

Tissue engineering, long a matter of myth and dream throughout the history of medicine, is now a practical reality. A wide spectrum of biological materials are used in the field of urology to treat disease and to overcome human disabilities, including tissue grafts and organ transplantation. Laboratory-engineered bioproducts for the off-the-shelf replacement and reconstruction of tissue is now almost at hand. This article presents a glimpse into the past by highlighting a number of early pioneering works in the field of tissue transplantation and cell culture technologies.

Biocompatible Materials↗

PubMed1999

Gene-enhanced tissue engineering: applications for bone healing using cultured periosteal cells transduced retrovirally with the BMP-7 gene.

Periosteum has cell populations, including osteoprogenitor and chondroprogenitor cells, that can be grown in cell culture and form both bone and cartilage under appropriate conditions. The authors have shown previously that cultured periosteal cells can be used in the tissue engineering of bone, and they demonstrated substantial bone formation in a rabbit cranial defect model. In the current study, principles of tissue engineering were combined with principles of gene therapy to produce cultured periosteal cells transduced retrovirally with the bone morphogenetic protein 7 (BMP-7) gene to be used in the treatment of bone defects. Human BMP-7 complementary deoxyribonucleic acid was generated from a cell line using reverse transcription polymerase chain reaction and cloned into a retroviral vector plasmid. Retroviral vector particles were then used to transduce New Zealand White rabbit periosteal cells. Transduced periosteal cells demonstrated substantial production of both BMP-7 messenger ribonucleic acid by Northern blot analysis and BMP-7 protein by enzyme-linked immunosorbent assay. These cells were then seeded into polyglycolic acid (PGA) matrices and used to repair critical-size rabbit cranial defects. At 12 weeks, defect sites repaired with BMP-7-transduced periosteal cells/PGA had significantly increased radiographic and histological evidence of bone repair compared with those defect sites repaired with negative control-transduced cells/PGA, nontransduced cells/PGA, PGA alone, or unrepaired defects. Thus, this study demonstrates successfully a tissue engineering approach to bone repair using genetically modified cells.

Animals↗

PubMed2004

Bioartificial tracheal grafts: can tissue engineering keep its promise?

A huge variety of graft materials and transplantation approaches have been applied for decades in order to generate a clinically applicable tracheal substitute; so far, without success. Today, tissue engineering, the creation of man-made functional biological organs or tissue replacements from biodegradable carrier structures and autologous cells, may represent an alternative to the shortage of suitable grafts for reconstructive airway surgery. Partial success has been obtained by numerous groups following different concepts and strategies. In this article, tissue engineering approaches towards the bioartificial airway prosthesis are discussed, focusing primarily on recent developments in the field.

Animals↗

PubMed2006

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion: a continuum model of matrix metabolism and transport.

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue- engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

Biological Transport↗

PubMedDate not supplied

Tissue engineering of bone: the reconstructive surgeon's point of view.

Bone defects represent a medical and socioeconomic challenge. Different types of biomaterials are applied for reconstructive indications and receive rising interest. However, autologous bone grafts are still considered as the gold standard for reconstruction of extended bone defects. The generation of bioartificial bone tissues may help to overcome the problems related to donor site morbidity and size limitations. Tissue engineering is, according to its historic definition, an "interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function". It is based on the understanding of tissue formation and regeneration and aims to rather grow new functional tissues than to build new spare parts. While reconstruction of small to moderate sized bone defects using engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of large-volume defects remains challenging. Therefore vascularization concepts gain on interest and the combination of tissue engineering approaches with flap prefabrication techniques may eventually allow application of bone-tissue substitutes grown in vivo with the advantage of minimal donor site morbidity as compared to conventional vascularized bone grafts. The scope of this review is the introduction of basic principles and different components of engineered bioartificial bone tissues with a strong focus on clinical applications in reconstructive surgery. Concepts for the induction of axial vascularization in engineered bone tissues as well as potential clinical applications are discussed in detail.

Bioartificial Organs↗

PubMed2005

Tissue-engineering approaches for axonal guidance.

Owing to the profound impact of nervous system damage, extensive studies have been carried out aimed at facilitating axonal regeneration following injury. Tissue engineering, as an emerging and rapidly growing field, has received extensive attention for nervous system axonal guidance. Numerous engineered substrates containing oriented extracellular matrix molecules, cells or channels have displayed potential of supporting axonal regeneration and functional recovery. Most attempts are focused on seeking new biomaterials, new cell sources, as well as novel designs of tissue-engineered neuronal bridging devices, to generate safer and more efficacious neuronal tissue repairs.

Animals↗

PubMed2005

Cell proliferation rates in an artificial tissue-engineered environment.

Worldwide, and particularly in Europe, Japan and the USA, cardiovascular disease is a major killer. It can be treated using tissue or organ transplant surgery, but donor organs may be scarce. Tissue engineering is the integration of engineering principles and biology to produce satisfactory synthetic replacement body parts, using viable cells in a suitable matrix, for regenerative medicine. The aim of this study was to measure and compare cell proliferation kinetics after different time intervals of myofibroblasts in a synthetic matrix, thus to be able to deduce the period that a transplanted-cell population can be expected to survive in a tissue-engineered environment. Porcine aortic wall cells were grown in a porous sponge scaffold, that later could be fashioned into aortic or heart valve substitutes. Freshly acquired cells were seeded on identical sponges and were grown under normal culture conditions for a period of 4 weeks. Seeding concentration was a million cells per sponge. Cells progressively populated the sponges, both covering the surface and infiltrating the depth of the matrix, via sponge pores. Samples were taken at 1 week and at 4 weeks, and the rate of cell proliferation was determined by the metaphase arrest technique. Specimens were also taken for light and electron microscopy to determine whether these transplanted cells were capable of synthesizing their own extracellular matrix.

Algorithms↗

PubMed2003

Effect of basic fibroblast growth factor on vascularization in esophagus tissue engineering.

We carried out an experimental study to evaluate the effect of basic fibroblast growth factor (bFGF)-containing collagen gel on vascularization in esophageal tissue engineering. We compared an acellular collagen sponge scaffold and an acellular collagen gel scaffold in combination with bFGF using a canine model. The construct was implanted in the cervical esophagus and the regenerated tissue was evaluated one month after surgery. Histological analysis confirmed a significantly large amount of blood vessels in the bFGF-containing collagen gel group as compared to the collagen gel group without bFGF (bFGF (-)). However, in the collagen sponge groups, no difference was observed between the bFGF (+) group and the bFGF (-) group. These results showed that bFGF-containing collagen gel is suitable not only for an acellular scaffold for tissue engineering but also for an effective tropic factor vehicle in vivo.

Animals↗

PubMed2004

Animal models for intestinal tissue engineering.

Although total parenteral nutrition prevents patients with short bowel syndrome from dying of starvation, having short bowel remains a severely debilitating condition. The best current treatment for inadequate absorptive surface area is through intestinal transplantation. However, this therapy is associated with significant morbidity and patients suffer from consequences of long-term immunosuppression. Additionally, the numbers of organs are limited. A new frontier in medicine is the field of tissue engineering. We will review the progress of intestinal bioengineering with a focus on the use of animal models. Investigators initially used autologous tissue as a patch to study intestinal regeneration. Subsequent studies focused on the use of absorbable biomaterials as a patch for tissue ingrowth. The most novel methodology consists of seeding a resorbable scaffold and implanting this construct to observe the regeneration of neointestine. Successful creation of esophagus, stomach, small bowel and colon has been demonstrated. Although these studies are preliminary, the results suggest that tissue-engineered intestine will become a real therapeutic option in the not too distant future for patients with inadequate intestinal tissue.

Absorbable Implants↗

PubMed2003

Chitosan-thioglycolic acid conjugate: a new scaffold material for tissue engineering?

It was the aim of this study to evaluate chitosan-thioglycolic acid (chitosan-TGA) conjugate as scaffold material in tissue engineering. Chitosan was modified by the introduction of thiol groups. Briefly, TGA was introduced to chitosan via amide bond formation mediated by a carbodiimide. The properties of the resulting polymer were thereby altered in regard to water solubility, mucoadhesion, biodegradability and in situ gelling compared to the original polymer. Due to the immobilised thiol groups (240+/-30 micromol thiol groups per gram polymer), the viscosity of a 1.5% chitosan-TGA solution was improved 4.3-fold. This can be explained by the formation of disulphide bonds within this polymeric network. The conjugate was tested as scaffold material in form of a gel and sheets. Furthermore, the influence of the thiol groups on the viability of L-929 mouse fibroblasts was evaluated. It was shown that the L-929 mouse fibroblasts grew on both scaffolds despite the thiol groups, although the different surface conditions seemed to have an influence on the growing rate. Chitosan-TGA sheets seemed to be the more preferred layer. The improved in situ gelling may be important for ongoing developments. Direct injectable matrices at the site of tissue damage mimicking the tissue being restored may be a future trend on this topic. Hence, chitosan-TGA is a promising candidate as scaffold material in tissue engineering.

Animals↗

PubMed2005

Laser surface modification of poly(epsilon-caprolactone) (PCL) membrane for tissue engineering applications.

Ultra-thin polycaprolactone (PCL) produced by bi-axial stretching was previously shown to have significant advantage for membrane tissue engineering. However, the permeability of the membrane needs to be enhanced. In this study, ablation experiments using femtosecond laser and excimer laser were carried out to modify the PCL surface. The use of the femtosecond laser produces neat drilled-through holes while the excimer laser is employed to produce blind-holes on the membrane. The modified surface of the membrane was studied and analyzed for different laser parameters (such as pulse energy and pulse repetition rate and characterized using several techniques that include optical microscopy, scanning electron microscopy and water contact angle measurements). Results showed that the morphological surface changes with different laser parameters, and the water contact angle decreases as the surface of the membrane is modified. The decrease in water contact angle suggests that surface of the membrane had become more hydrophilic than the non-laser treated membrane. The present study demonstrated that laser surface modification on the PCL can be achieved with high degree of success and precision. This paved the way for further enhancement in membrane tissue engineering.

Biocompatible Materials↗