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PubMedDate not supplied

Composite articular cartilage engineered on a chondrocyte-seeded aliphatic polyurethane sponge.

To circumvent the reconstructive disadvantages inherent in resorbable polyglycolic acid (PGA)/polylactic acid (PLA) used in cartilage engineering, a nonresorbable, and nonreactive polyurethane sponge (Tecoflex sponge, TS) was studied as both a cell delivery device and as an internal support scaffolding. The in vitro viability and proliferation of porcine articular chondrocytes (PACs) in TS, and the in vivo generation of new articular cartilage and long-term resorption, were examined. The initial cell attachment rate was 40%, and cell density increased more than 5-fold after 12 days of culture in vitro. PAC-loaded TS blocks were implanted into nude mice, became opalescent, and resembled native cartilage at weeks 12 and 24 postimplantation. The mass and volume of newly formed cartilage were not significantly different at week 24 from samples harvested at week 6 or week 12. Safranin O-fast green staining revealed that the specimens from cell-loaded TS groups at week 12 and week 24 consisted of mature cartilage. Collagen typing revealed that type II collagen was present in all groups of tissue-engineered cartilage. In conclusion, the implantation of PAC-TS resulted in composite tissue-engineered articular cartilage with TS as an internal support. Long-term observation (24 weeks) of mass and volume showed no evidence of resorption.

Animals↗
PubMedDate not supplied

Electrospun nanofiber fabrication as synthetic extracellular matrix and its potential for vascular tissue engineering.

Substantial effort is being invested by the bioengineering community to develop biodegradable polymer scaffolds suitable for tissue-engineering applications. An ideal scaffold should mimic the structural and purposeful profile of materials found in the natural extracellular matrix (ECM) architecture. To accomplish this goal, poly (L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (75:25) copolymer with a novel architecture produced by an electrospinning process has been developed for tissue-engineering applications. The diameter of this electrospun P(LLA-CL) fiber ranges from 400 to 800 nm, which mimicks the nanoscale dimension of native ECM. The mechanical properties of this structure are comparable to those of human coronary artery. To evaluate the feasibility of using this nanofibrous scaffold as a synthetic extracellular matrix for culturing human smooth muscle cells and endothelial cells, these two types of cells were seeded on the scaffold for 7 days. The data from scanning electron microscopy, immunohistochemical examination, laser scanning confocal microscopy, and a cell proliferation assay suggested that this electrospun nanofibrous scaffold is capable of supporting cell attachment and proliferation. Smooth muscle cells and endothelial cells seeded on this scaffold tend to maintain their phenotypic shape. They were also found to integrate with the nanofibers to form a three-dimensional cellular network. These results indicate a favorable interaction between this synthetic nanofibrous scaffold with the two types of cells and suggest its potential application in tissue engineering a blood vessel substitute.

Biocompatible Materials↗
PubMedDate not supplied

Novel pulse duplicating bioreactor system for tissue-engineered vascular construct.

Cell culture in a biomimetic environment is known to improve the mechanical endurance of tissue-engineered cardiovascular components. Our goal was to generate a bioreactor that can reproduce a wide range of pulsatile flows with a completely physiological pressure profile. The morphology and biochemical properties of tissue-engineered products were also studied to test the usefulness of this novel bioreactor. The combination of an outflow valve, compliance chamber, and resistant clamps together with a balloon pumping system was able to successfully reproduce both physiological systolic and diastolic pressures. The compliance chamber was especially effective in transforming the original peaky pressure waveform into a physiological pressure profile. The tissues, cultured under a physiological pressure waveform with pulsatile flow, presented widely distributed cells in close contact with each other. They also showed significantly higher cell numbers, total protein content, and proteoglycan-glycosaminoglycan content than cultured tissues under a peaky pressure wave or under static conditions. This new bioreactor system is suitable for evaluating a favorable environment for tissue-engineered cardiovascular components.

Animals↗
PubMedDate not supplied

Efficacy of engineered liver tissue based on poly-L-lactic acid scaffolds and fetal mouse liver cells cultured with oncostatin M, nicotinamide, and dimethyl sulfoxide.

To assess the feasibility of liver tissue equivalents based on selective propagation and differentiation of hepatocyte progenitors in three-dimensional (3D) culture, the efficacy of fetal mouse liver cells cultured in poly-L-lactic acid (PLLA) scaffolds in the presence of nicotinamide, dimethyl sulfoxide, and oncostatin M was investigated both in vitro and in vivo. The albumin production of PLLA-cultured fetal mouse liver cells in the presence of these three factors was remarkably enhanced with culture time, and after 4 weeks it attained almost the same production found in adult mouse hepatocytes cultured for 3 days in PLLA scaffolds, based on the unit DNA amount. In addition, implantation of engineered liver tissue based on this in vitro PLLA culture system into the peritoneal cavity of 70% hepatectomized mice showed a remarkably higher presence of albumin-positive engrafted cells 15 days after the operation when compared with fetal mouse liver cells or adult mouse hepatocytes freshly isolated and cultured for 1 day. These results demonstrate that the basic concept regarding the engineering of liver tissue equivalents based on in vitro selective propagation and differentiation of hepatocyte progenitors in 3D biodegradable scaffolds shows promise for future liver tissue engineering.

Albumins↗
PubMedDate not supplied

Seeding techniques and scaffolding choice for tissue engineering of the temporomandibular joint disk.

Temporomandibular joint (TMJ) disk removal, or diskectomy, is a detrimental yet necessary surgery for patients with extremely displaced disks. Tissue engineering is an enticing methodology for improvement of the postoperative outcome of diskectomy. Unfortunately, the field of tissue engineering of the TMJ disk is only in the early stages of development. The initial objective of this investigation was to study the cellular response of TMJ disk cells in alginate culture. However, a marked decrease in cell population and the lack of detection of extracellular matrix (ECM) products did not support the use of alginate culture. The second objective was then to attempt TMJ disk cell culture in polyglycolic acid (PGA) nonwoven meshes. However, as suitable seeding methods for TMJ disk cells on PGA had not been determined, three techniques were selected for study: spinner flask, orbital shaker, and a novel pelleting technique. PGA constructs maintained cellularity throughout the culture period, and scaffolds seeded with the spinner flask produced about 35 microg of collagen per construct. Thus, as evidenced by the production of a major extracellular component, PGA nonwoven meshes seeded with TMJ disk cells, using a spinner flask, may be a first positive culturing step in tissue engineering the TMJ disk.

Alginates↗
PubMedDate not supplied

Assessment of tissue ingrowth rates in polyurethane scaffolds for tissue engineering.

The continuous development of new biomaterials for tissue engineering and the enhancement of tissue ingrowth into existing scaffolds, using growth factors, create the necessity for developing adequate tools to assess tissue ingrowth rates into porous biomaterials. Current histomorphometric techniques evaluating rates of tissue ingrowth tend either to measure the overall tissue content in an entire sample or to depend on the user to indicate a front of tissue ingrowth. Neither method is particularly suitable for the assessment of tissue ingrowth rates, as these methods either lack the sensitivity required or are problematic when there is a tissue ingrowth gradient rather than an obvious tissue ingrowth front. This study describes a histomorphometric method that requires little observer input, is sensitive, and renders detailed information for the assessment of tissue ingrowth rates into porous biomaterials. This is achieved by examining a number of computer-defined concentric zones, which are based on the distance of a pixel from the scaffold edge. Each zone is automatically analyzed for tissue content, eliminating the need for user definition of a tissue ingrowth front and thus reducing errors and observer dependence. Tissue ingrowth rates in two biodegradable polyurethane scaffolds (Estane and polycaprolactone-polyurethane [PCLPU]) specifically designed for tissue engineering of the knee meniscus were assessed. Samples were subcutaneously implanted in rats with follow-up until 6 months. Especially at the earlier follow-up points, PCLPU scaffolds showed significantly higher tissue ingrowth rates than Estane scaffolds, making the PCLPU scaffold a promising candidate for further studies investigating meniscus tissue engineering.

Animals↗
PubMed1990

[The determination of trace amounts of protein in solutions containing surface-active substances].

A rapid and sensitive method for protein determination (0.5-16 micrograms) in samples of any volume containing various surfactants in concentration up to 1% is suggested. The method includes the protein acid denaturation, the solution of acid insoluble precipitate of detergent in ethanol (25-30%), the protein determination on nitrocellulose filter, dyeing by aminoblack 10 B, elution of dyed complex and colorimetric determination at 630 nm.

Amido Black↗
PubMedDate not supplied

Effects of in vitro preculture on in vivo development of human engineered cartilage in an ectopic model.

We investigated whether, and under which conditions (i.e., cell-seeding density, medium supplements), in vitro preculture enhances in vivo development of human engineered cartilage in an ectopic nude mouse model. Monolayer-expanded adult human articular chondrocytes (AHACs) were seeded into Hyalograft C disks at 1.3 x 10(7) cells/cm3 (low density) or 7.6 x 10(7) cells/cm3 (high density). Constructs were directly implanted subcutaneously in nude mice for up to 8 weeks or precultured for 2 weeks before implantation. Preculture medium contained either transforming growth factor-beta1 (TGF-beta1, 1 ng/mL), fibroblast growth factor-2, and platelet-derived growth factor (proliferating medium) or TGF-beta1 (10 ng/mL) and insulin (differentiating medium). Both in vitro and after in vivo implantation, constructs derived by cell seeding at high versus low density and precultured in differentiating versus proliferating medium generated more cartilaginous tissues containing higher amounts of glycosaminoglycan and collagen type II and lower amounts of collagen type I, and with higher equilibrium moduli. As compared with direct implantation of freshly seeded scaffolds, preculture of AHAC-Hyalograft C constructs in differentiating medium, but not in proliferating medium, supported enhanced in vivo development of engineered cartilage. The effect of preculture was more pronounced when constructs were seeded at low density as compared with high density. This study indicates that preculture of human engineered cartilage in differentiating medium has the potential to provide grafts with higher equilibrium moduli and enhanced in vivo developmental capacity than freshly seeded scaffolds. These findings need to be validated in an orthotopic model system.

Animals↗
PubMedDate not supplied

Novel methodology for fabrication of tissue-engineered tubular constructs using magnetite nanoparticles and magnetic force.

Novel technologies for creating three-dimensional constructs with complex shapes would be highly useful in tissue engineering. In the present study, tubular structures were constructed using magnetic force. Magnetite nanoparticles in cationic liposomes were taken up by target cells. The magnetically labeled cells were seeded onto ultralow-attachment plates, and a magnet was placed under the wells. After 24 h of culture, the magnetically labeled cells formed a cell sheet. Subsequently, when a cylindrical magnet was rolled onto the cell sheet, the cell sheet was attracted to the magnet and formed a tube around it. The magnet was then removed, leaving behind a tubular structure. Two types of tissue were used to create tubular structures: urinary tissue, consisting of a monotypic urothelial cell layer; and vascular tissue, consisting of heterotypic layers of endothelial cells, smooth muscle cells, and fibroblasts. The present results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach to constructing tissue-engineered tubular structures.

Animals↗
PubMedDate not supplied

Fabrication and endothelialization of collagen-blended biodegradable polymer nanofibers: potential vascular graft for blood vessel tissue engineering.

Electrospun collagen-blended poly(L-lactic acid)-co-poly(epsilon-caprolactone) [P(LLA-CL), 70:30] nanofiber may have great potential application in tissue engineering because it mimicks the extracellular matrix (ECM) both morphologically and chemically. Blended nanofibers with various weight ratios of polymer to collagen were fabricated by electrospinning. The appearance of the blended nanofibers was investigated by scanning electron microscopy and transmission electron microscopy. The nanofibers exhibited a smooth surface and a narrow diameter distribution, with 60% of the nanofibers having diameters between 100 and 200 nm. Attenuated total reflectance-Fourier transform infrared spectra and X-ray photoelectron spectroscopy verified the existence of collagen molecules on the surface of nanofibers. Human coronary artery endothelial cells (HCAECs) were seeded onto the blended nanofibers for viability, morphogenesis, attachment, and phenotypic studies. Five characteristic endothelial cell (EC) markers, including four types of cell adhesion molecule and one EC-preferential gene (von Willebrand factor), were studied by reverse transcription-polymerase chain reaction. Results showed that the collagen-blended polymer nanofibers could enhance the viability, spreading, and attachment of HCAECs and, moreover, preserve the EC phenotype. The blending electrospinning technique shows potential in refining the composition of polymer nanofibers by adding various ingredients (e.g., growth factors) according to cell types to fabricate tissue-engineering scaffold, particularly blood vessel-engineering scaffold.

Biocompatible Materials↗
PubMedDate not supplied

Adipose tissue engineering based on mesenchymal stem cells and basic fibroblast growth factor in vitro.

Despite the clinical need for reconstructive and plastic surgery, the supply of engineered adipose tissue equivalents still remains a challenge. As yet, only preadipocytes have been applied as a cell material for the in vitro tissue engineering of fat. Herein, we report the establishment of a three-dimensional (3-D) long-term cell culture, using bone marrow-derived mesenchymal stem cells (MSCs) as an alternative cell source and custom-made poly(lactic-co-glycolic acid) (PLGA) scaffolds as a cell carrier. Cell-polymer constructs were cultivated for 4 weeks in both the absence and presence of basic fibroblast growth factor (bFGF), which was previously shown to strongly enhance the adipogenesis of MSCs in conventional 2-D short-term culture. A striking enhancement of the adipogenic differentiation of MSCs and tissue development caused by bFGF in the 3-D culture was observed by osmium tetroxide histology and scanning electron microscopy. At the molecular level, reflecting the increased accumulation of lipids, bFGF increased the enzymatic activity of glycerol-3-phosphate dehydrogenase, a late marker of adipogenesis, and the expression of adipocyte-specific genes peroxisome proliferator activated receptor-gamma2 (PPARgamma2) and glucose transporter-4 (GLUT4), as assessed by reverse transcription-polymerase chain reaction. This study demonstrates that the use of bone marrow-derived MSCs, especially in combination with bFGF, may represent a promising approach to adipose tissue engineering.

Adipogenesis↗
PubMedDate not supplied

Bioresponsive phosphoester hydrogels for bone tissue engineering.

Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.

Alkaline Phosphatase↗
PubMedDate not supplied

Quantitative outcome measures of cartilage repair in patients treated by tissue engineering.

Reliable and reproducible outcome measures are essential to assess the efficacy of competing and novel tissue-engineering techniques. The aim of this study was to compare traditional histological analyses with newly developed quantitative biochemical outcome measures for the repair of articular cartilage. The production of a new anti-peptide antibody and the development and validation of a novel method for the extraction and immunoassay of type I collagen are described. The assay was used, in conjunction with existing assays for type II collagen and proteoglycans, to measure levels of the matrix components in repair tissue biopsies obtained from patients treated with the new tissue-engineering therapy Hyalograft C. Frozen sections cut from the same biopsies were stained for proteoglycans, using safranin O, and immunohistochemical analysis was used to assess type I and II collagen staining. Although there was general agreement between the extent of staining and the amounts of the three matrix components, there was a large degree of overlap in biochemical content between biopsies classified histologically on the basis of low, moderate, or abundant staining. The results demonstrate that histological grading of matrix protein abundance to classify repair cartilage as hyaline or fibrocartilagenous is often misleading. In addition, we demonstrate for the first time the ability to measure collagen cross-links in repair tissue biopsies and show that it can be used as a surrogate marker for tissue maturity. Our new range of biochemical techniques provides a standardized method to assess the quality of both engineered cartilage produced in vitro and repair tissue biopsies obtained after in vivo implantation.

Animals↗
PubMedDate not supplied

Engineered adipose tissue from human mesenchymal stem cells maintains predefined shape and dimension: implications in soft tissue augmentation and reconstruction.

Soft tissue augmentation is a widespread practice in plastic and reconstructive surgery. The objective of the present study was to engineer adipose tissue constructs with predefined shape and dimensions, potentially utilizable in soft tissue augmentation and reconstruction, by encapsulating adult stem cell-derived adipogenic cells in a biocompatible hydrogel system. Bone marrow-derived adult human mesenchymal stem cells (hMSCs) were preconditioned by 1 week of exposure to adipogenic- inducing supplement followed by photoencapsulation in poly(ethylene glycol) diacrylate (PEGDA) hydrogel in predefined shape and dimensions. In two parallel experiments, the resulting hMSC-derived adipogenic cell-polymer constructs were either incubated in vitro in adipogenic medium or implanted in vivo in the dorsum of immunodeficient mice for 4 weeks. Tissue-engineered adipogenic constructs demonstrated positive reaction to oil red O staining both in vitro and in vivo, and expressed PPAR-gamma2 adipogenic gene marker in vivo. By contrast, control PEGDA hydrogel constructs encapsulating undifferentiated hMSCs failed to demonstrate the adipogenic gene marker and were negative for oil red O staining. Recovered in vitro and in vivo constructs maintained their predefined physical shape and dimensions. These data demonstrate that adipose tissue engineered from human mesenchymal stem cells can retain predefined shape and dimensions for soft tissue augmentation and reconstruction.

Adipose Tissue↗
PubMedDate not supplied

Biocompatible nanofiber matrices for the engineering of a dermal substitute for skin regeneration.

Natural and synthetic biodegradable nanofibers are extensively used for biomedical applications and tissue engineering. Biocompatibility and a well-established safety profile for polycaprolactone (PCL) and collagen represent a favorable matrix for preparing a dermal substitute for engineering skin. Collagen synthesized by fibroblasts is a good surface active agent and demonstrates its ability to penetrate a lipid-free interface. During granulation tissue formation, fibronectin provides a temporary substratum for migration and proliferation of cells and provides a template for collagen deposition, which increases stiffness and tensile strength of this healing tissues. The objective of this study was to fabricate nanofiber matrices from novel biodegradable PCL and collagen to mimic natural extracellular matrix (ECM) and to examine the cell behavior, cell attachment, and interaction between cells and nanofiber matrices. Collagen nanofiber matrices show a significant (p < 0.001) level of fibroblast proliferation and increase up to 54% compared with control tissue culture plate (TCP) after 72 h. The present investigation shows that PCL-coated collagen matrices are suitable for fibroblast growth, proliferation, and migration inside the matrices. This novel biodegradable PCL and collagen nanofiber matrices support the attachment and proliferation of human dermal fibroblasts and might have potential in tissue engineering as a dermal substitute for skin regeneration.

Biocompatible Materials↗
PubMedDate not supplied

In vivo response of polylactic acid-alginate scaffolds and bone marrow-derived cells for cartilage tissue engineering.

Successful application of tissue-engineering techniques to damaged biological structures is determined by functional performance in vivo. This study evaluated the in vivo response of a tissue-engineered construct composed of a polylactic acid-alginate amalgam seeded with bone marrow-derived mesenchymal stem cells and stimulated in vitro with transforming growth factor beta for cartilage tissue engineering. Constructs were placed in cylindrical osteochondral defects in the canine femoral condyle and examined 6 weeks postoperatively by gross, histological, immunohistochemical, and biomechanical analyses. In the course of 6 weeks in vivo, the defects filled with a cartilaginous tissue regardless of whether cell-seeded (experimental) or cell-free (control) constructs were implanted; however, the quality of the tissue differed between the experimental and control defects. Cell-seeded experimental defects showed more cartilage-like matrix quality, cell distribution, and proteoglycan staining. Biomechanically, experimental and control specimens exhibited similar behavior; however, both tissues were still immature compared with normal cartilage. The evidence accumulated in this study showed a modest acceleration of the in vivo healing of cell-seeded constructs but also demonstrated a reparative response of cell-free constructs. This finding suggests that the constructs prepared from the PLA-alginate amalgam may serve as a means for host cell attachment.

Alginates↗
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↗