New approaches to tissue engineering aim to exploit endogenous strategies such as those occurring in prenatal development and recapitulated during postnatal healing. Defining tissue template specifications to mimic the environment of the condensed mesenchyme during development allows for exploitation of tissue scaffolds as delivery devices for extrinsic cues, including biochemical and mechanical signals, to drive the fate of mesenchymal stem cells seeded within. Although a variety of biochemical signals that modulate stem cell fate have been identified, the mechanical signals conducive to guiding pluripotent cells toward specific lineages are less well characterized. Furthermore, not only is spatial and temporal control of mechanical stimuli to cells challenging, but also tissue template geometries vary with time due to tissue ingrowth and/or scaffold degradation. Hence, a case study was carried out to analyze flow regimes in a testbed scaffold as a first step toward optimizing scaffold architecture. A pressure gradient was applied to produce local (nm-micron) flow fields conducive to migration, adhesion, proliferation, and differentiation of cells seeded within, as well as global flow parameters (micron-mm), including flow velocity and permeability, to enhance directed cell infiltration and augment mass transport. Iterative occlusion of flow channel dimensions was carried out to predict virtually the effect of temporal geometric variation (e.g., due to tissue development and growth) on delivery of local and global mechanical signals. Thereafter, insights from the case study were generalized to present an optimization scheme for future development of scaffolds to be implemented in vitro or in vivo. Although it is likely that manufacture and testing will be required to finalize design specifications, it is expected that the use of the rational design optimization will reduce the number of iterations required to determine final prototype geometries and flow conditions. As the range of mechanical signals conducive to guiding cell fate in situ is further elucidated, these refined design criteria can be integrated into the general optimization rubric, providing a technological platform to exploit nature's endogenous tissue engineering strategies for targeted tissue generation in the lab or the clinic.

Synthetic urothelium is an important goal for the tissue-engineering field that would have great utility for treating diseases and congenital defects affecting the urinary tract. A key step in the development of synthetic tissue is optimizing the conditions for coating biomaterials with cells of interest. Initial cell attachment is an important consideration when designing tissue-engineering scaffolds. The scaffold environment must also be conducive to cell proliferation and differentiation. The most popular materials for tissue-engineering scaffold often have suboptimal properties when analyzed for cell attachment and growth. It would then be of interest to know, for urinary tract tissue-engineering applications, which extracellular matrix protein coatings can facilitate urothelial cell attachment and encourage growth. Cells grown on 96-well cycloolefin plates coated with type IV or type I collagen exhibited improved initial attachment over plates coated with fibronectin or laminin. After 20 h, deoxyribonucleic acid synthesis was found to increase in cultures grown on type IV collagen, fibronectin, and laminin. Total metabolic activity of urothelial cell cultures was also monitored, and no difference was seen between any protein-coating conditions. The development of such reliable assays will be beneficial in monitoring the fate of scaffolds seeded with human urothelial cells.

Cell-based regenerative medicine, including tissue engineering, is a novel approach to reconstituting tissues that do not spontaneously heal, such as damaged cartilage, and to curing diseases caused by malfunctioning cells. Typically, manufacturing processes to generate cartilage for replacement therapies involve isolation and expansion of cells from cartilage biopsies. A challenge in the field is potential contamination by other cell types (e.g., fibroblast-like cells), which can overgrow the desired cells during culturing and may ultimately compromise clinical efficacy. No standard analytical system has been absolutely effective in ensuring the identity of these cell-based products. Therefore, we tested deoxyribonucleic acid methylation analysis as a quality assessment tool, applying it to Genzyme's Carticel product, a chondrocyte implant that the Food and Drug Administration has approved. We identified 7 potent discriminators by assaying candidate genomic regions derived from methylation discovery approaches and literature searches regarding a functional role of genes in chondrocyte biology. Using a support vector machine, we trained an optimal cell type classifier that was absolutely effective in discriminating chondrocytes from synovial membrane derived cells, the major potential contaminant of chondrocyte cultures. The abundant marker availability and high quality of this assay format also suggest it as a potential quality control test for other cell types grown or manipulated in vitro.

The development of minimally invasive therapeutics for orthopedic clinical conditions has substantial benefits, especially for osteoporotic fragility fractures and vertebral compression fractures. Poly(ester urethane)urea (PEUUR) foams are potentially useful for addressing these conditions because they cure in situ upon injection to form porous scaffolds. In this study, the effects of water concentration and polyester triol composition on the physicochemical, mechanical, and biological properties of PEUUR foams were investigated. A liquid resin (lysine diisocyanate) and hardener (poly(epsilon-caprolactone-co-glycolide-co-DL-lactide) triol, tertiary amine catalyst, anionic stabilizer, and fatty acid-derived pore opener) were mixed, and the resulting reactive liquid mixture was injected into a mold to harden. By varying the water content over the range of 0.5 to 2.75 parts per hundred parts polyol, materials with porosities ranging from 89.1 to 95.8 vol-% were prepared. Cells permeated the PEUUR foams after 21 days post-seeding, implying that the pores are open and interconnected. In vitro, the materials yielded non-cytotoxic decomposition products, and differences in the half-life of the polyester triol component translated to differences in the PEUUR foam degradation rates. We anticipate that PEUUR foams will present compelling opportunities for the design of new tissue-engineered scaffolds and delivery systems because of their favorable biological and physical properties.

The surface modification of islets using poly(ethylene glycol) (PEG) is being studied as a means of preventing host immune responses against transplanted islets. In this study, to completely shield islets with PEG molecules, we increased the amount of PEG conjugated to islet surfaces, by multiple PEGylation or amplified PEGylation using poly-L-lysine, poly(allylamine), or poly(ethyleneimine), respectively. Amplified PEGylation was associated with islet cytotoxicity and functional impairment, but multiple PEGylation affected neither islet viability nor functionality. In addition, when triply PEGylated islets were allotransplanted into diabetic recipients, these islets survived in 3 of the 7 recipients for more than 100 days without any immunosuppressive treatment. Moreover, the blood glucose levels of these 3 recipients were stable and in the normal range. Immunohistochemical analysis showed that 3 of 7 triply PEGylated islets transplants survived for 100 days and that 4 that were rejected before day 20 were all immunologically protected from immune cells. However, unmodified islets were completely destroyed within 1 week. Consequently, we suggest that multiple PEGylation offers an effective means of reducing the immunogenicity of transplanted islets by increasing the amount of surface-bound PEG.

Cell-based bone tissue engineering strategies have been effectively applied toward the development of grafting templates for skeletal repair and regeneration, but remain limited by inadequate availability of a robust mineralizing cell source. Dermal fibroblasts have emerged as a particularly promising cell alternative because they are harvested from autologous donors through minimally invasive skin biopsy and display a high capacity for in vitro expansion. In the present study, we investigated retroviral gene delivery of the osteogenic transcription factor Runx2 as a mineralization induction strategy in primary dermal fibroblasts. We demonstrate that constitutive overexpression of Runx2 induced osteogenic gene expression and mineralized nodule deposition in fibroblasts cultured on 3-dimensional fibrous collagen disks in vitro. Fourier transform infrared analysis revealed that Runx2 expressing fibroblasts deposit a carbonate-containing, poorly crystalline hydroxyapatite, whereas control constructs did not contain biologically-equivalent mineral. Importantly, Runx2-transduced fibroblasts formed mineralized templates in vivo after implantation in a subcutaneous, heterotopic site, whereas minimal mineralization was evident in control constructs. Furthermore, immunohistochemical analysis indicated that Runx2-engineered cells co-localized with mineral deposits in vivo, suggesting that nodule formation primarily originated from transplanted donor cells. These results establish Runx2-genetic engineering as a strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with compromised ability to recruit endogenous osteoprogenitors to the site of injury as a result of extreme trauma, age, radiation treatment, or osteolytic disease.

Large and retracted rotator cuff tendon tears fail to repair or retear after surgical intervention. This study attempted to develop novel tissue-engineering approaches using tenocyte-seeded bioscaffolds for tendon reconstruction of massive rotator cuff tendon defect in rabbits. Porcine small intestine submucosa (Restore) and type I/III collagen bioscaffold (ACI-Maix) were chosen as bioscaffold carriers for autologous tenocytes. Biological characterization of autologous tenocytes was conducted before the implantation. The tenocyte-seeded bioscaffolds were implanted as interposition grafts to reconstruct massive rotator cuff tendon defects in rabbits. In situ reimplantation of the autologous rotator cuff tendon, excised during defect creation, served as a positive control. Histological outcomes were analyzed and semi-quantitatively graded at 4 and 8 weeks after surgery. At 4 weeks, both tenocyte-seeded bioscaffolds displayed inflammatory reaction similar to bioscaffold-only cuff reconstruction, and the histological grading were inferior to control repair. However, at 8 weeks, inflammatory reaction of both tenocyte-seeded bioscaffolds were dramatically less than with bioscaffold alone. In addition, bioscaffolds seeded with tenocytes generated a histological appearance similar to that of the positive control. The implantation of autologous tenocytes on collagen-based bioscaffolds results in better rotator cuff tendon healing and remodeling than with the implantation of bioscaffold alone.

Tissue engineering offers a promising solution to the replacement of anterior cruciate ligament. A decellularized porcine patella tendon scaffold was produced by immersing whole tissues sequentially in hypotonic buffer, 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer, and nuclease solution prior to sterilization with 0.1% (w/v) peracetic acid. Initial studies revealed that primary human tenocytes would attach to, but failed to penetrate into, the decellularized scaffold. A novel use of ultrasonication was therefore developed to allow extrinsic cells to migrate into the acellular scaffold. Various intensities of ultrasonication were tested in order to produce a microscopically more open porous matrix without damaging the overall architecture of the scaffold. Ultrasonication treatment with the intensity of 360 W and a pulse time of 1 s for a total of 1 min was found to be the optimal treatment. This process did not have a significant effect upon the biochemical constituents (collagen, glycosaminoglycans), nor did it denature the collagen. Moreover, the acellular sonicated scaffold retained the essential biomechanical characteristics of the native tissue. Primary human tenocytes penetrated into the center of whole acellular sonicated scaffolds over a 3-week period in static culture. The viability of the cells in the center of the scaffold (depth of circa 2.5 mm) was, however, compromised. To circumvent the problem of nutrient limitation, acellular sonicated scaffolds were split into fascicular scaffolds (500 mum thick). Cells seeded onto the fascicular scaffolds penetrated throughout the scaffold and remained viable after 3 weeks of culture. This study has shown that an acellular biocompatible tendon scaffold can be produced using 0.1% (w/v) SDS and that ultrasonication can provide a novel method to enhance the recellularization of decellularized natural tissues.

The purpose of this study was to evaluate the use of adipose-derived stem cells (ADSCs) as a source for full-thickness cartilage repair in an animal model. Autologous ADSCs were isolated and induced with growth medium and placed in a fibrin glue scaffold and into 3-mm x 4-mm full-thickness chondral defects in rabbits with negative controls. Specimens were evaluated for early healing using immunostaining, Western blotting, reverse transcriptase polymerase chain reaction, transfection with the Lac Z gene, and quantitative assessment. Twelve of 12 (100%) articular surface defects containing tissue-engineered stem cell constructs healed with hyaline-like cartilage, versus 1 of 12 (8%) in the control group (p < .001). There was complete healing to subchondral bone in 12 of 12 experimental defects (100%), and 10 of 12 (83%) had seamless annealing to the native cartilage. Aggrecan, superficial zone protein, collagen type II messenger ribonucleic acid, and Lac-Z gene products were identified in 12 of 12 experimental specimens, which exhibited a collagen type II:I protein ratio similar to that of normal rabbit cartilage. Quantitative histologic analysis revealed an average score of 18.2 of 21 in the experimental group, compared with 10.0 in the controls (p = .001). Induced ADSCs supported in a fibrin glue matrix are a promising cell source for cartilage tissue engineering.

Ascorbic acid is known to influence proliferation and functional properties of several cell types and is therefore widely used in tissue engineering. In this study, the effect of ascorbic acid on the proliferation and functional properties of hyalocytes was evaluated. Hyalocytes were cultured with different amounts of ascorbic acid in classical two-dimensional (2-D) cultures and a three-dimensional (3-D) pellet culture system. Ascorbic acid enhanced hyalocyte proliferation dose-dependently at concentrations between 0.1 and 3 microg/mL; proliferation was constant over a wide concentration range up to 150 microg/mL, concentrations of 500 microg/mL showed toxic effects. In 2-D hyalocyte culture, the accumulation of glycosaminoglycans (GAG) and collagens increased in response to ascorbic acid supplementation of 10 or 200 microg/mL. Normalized to the cell number, GAG production was not influenced, whereas collagen production increased. These results could be verified in a pellet-like 3-D culture system. Ascorbic acid also influenced hyalocytes on the mRNA level; the expression of COL11A1 was clearly enhanced by ascorbic acid. To conclude, ascorbic acid modulates proliferation and collagen accumulation of hyalocytes; it also influences mRNA expression of the cells. Taken together with the fact that ascorbic acid is present in high concentrations in the vitreous body, this vitamin seems to be an important factor for in vitro hyalocyte culture.