An optimal strategy for the functional tissue engineering of articular cartilage, particularly to accelerate construct development, may incorporate sequential application of different growth factors and applied deformational loading.

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.

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Objective Chondrocyte-seeded agarose constructs of 4 mm diameter (2.34 mm thickness) develop spatially inhomogeneous material properties with stiffer outer edges and a softer central core suggesting nutrient diffusion limitations to the central construct region1. The effects of reducing construct thickness and creating channels running through the depth of the thick constructs were examined. Methods In Study 1, the properties of engineered cartilage of 0.78mm (thin) or 2.34mm (thick) thickness were compared. In Study 2, a single nutrient channel (1 mm diameter) was created in the middle of each thick construct. In Study 3, the effects of channels on larger 10 mm diameter, thick constructs was examined. Results Thin constructs developed superior mechanical and biochemical properties than thick constructs. The channeled constructs developed significantly higher mechanical properties versus control channel-free constructs while exhibiting similar GAG and collagen content. Collagen staining suggested that channels resulted in a more uniform fibrillar network. Improvements in constructs of 10mm diameter were similarly observed. Conclusions This study demonstrated that more homogeneous tissue engineered cartilage constructs with improved mechanical properties can be achieved by reducing their thickness or incorporating macroscopic nutrient channels. Our data further suggests that these macroscopic channels remain open long enough to promote this enhanced tissue development while exhibiting the potential to refill with cell elaborated matrix with additional culture time. Together with reports that <3 mm defects in cartilage heal in vivo and that irregular holes are associated with clinically used osteochondral graft procedures, we anticipate that a strategy of incorporating macroscopic channels may aid the development of clinically-relevant engineered cartilage with functional properties.

It was hypothesized that previously optimized serum-free culture conditions for juvenile bovine chondrocytes could be adapted to generate engineered cartilage with physiologic mechanical properties in a preclinical, adult canine model. Primary or passaged (using growth factors) adult chondrocytes from three adult dogs were encapsulated in agarose, and cultured in serum-free media with transforming growth factor-beta3. After 28 days in culture, engineered cartilage formed by primary chondrocytes exhibited only small increases in glycosaminoglycan content. However, all passaged chondrocytes on day 28 elaborated a cartilage matrix with compressive properties and glycosaminoglycan content in the range of native adult canine cartilage values. A preliminary biocompatibility study utilizing chondral and osteochondral constructs showed no gross or histological signs of rejection, with all implanted constructs showing excellent integration with surrounding cartilage and subchondral bone. This study demonstrates that adult canine chondrocytes can form a mechanically functional, biocompatible engineered cartilage tissue under optimized culture conditions. The encouraging findings of this work highlight the potential for tissue engineering strategies using adult chondrocytes in the clinical treatment of cartilage defects.

Unlike native cartilage explants that are used in autologous tissue transfer procedures, engineered cartilage constructs are typically highly fragile when first formed and must rely on cellular activity to develop over time. However, inflammatory cytokines such as interleukin-1alpha (IL-1alpha) are often present in target joints and may interfere with this development process. Herein we examine to what extent nascent engineered tissue is susceptible to chemical perturbations by IL-1alpha (10 ng/mL), especially when compared to native explants, and whether in vitro preconditioning may promote sufficient integrity to lessen this impact. The studies were carried out using a chemically defined medium supplemented with or without the antiinflammatory steroid dexamethasone. We find that engineered tissue (bovine chondrocytes in agarose hydrogel) at early time points (days 0 and 14) does not grow when exposed to the cytokine even temporarily, but both bovine explants and more developed engineered tissue (day 28) are able to withstand the same exposure without degradation of properties. We argue therefore that some in vitro preconditioning may be necessary to promote both sufficient mechanical integrity and the chemical fortitude without which insufficiently developed engineered constructs will not survive the harsh mechanochemical environment within the joint.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition with a birth incidence of 1/3,500. Around 50% of cases are due to new mutations. The NF1 gene maps to 17q11.2 and encodes neurofibromin. NF1 is a “classical” tumor suppressor gene. Congenital disseminated NF1 is rare with just two cases previously reported. We present a deceased baby with congenital disseminated NF1 in whom we performed molecular studies. A germline mutation (R461X) in exon 10a of the NF1 gene was found. A 2 bp deletion (3508delCA) in codon 1170 of exon 21 was identified in DNA derived from some tumor tissue. Loss of heterozygosity in NF1 and TP53 was observed in other tumor samples. No microsatellite instability was observed in the tumor samples. This is the first report of molecular analysis of the NF1 locus in a patient with disseminated congenital neurofibromatosis. This case had a de novo germline mutation in NF1 and three documented somatic mutations in the NF1 and TP53 genes in tumor specimens. © 2008 Wiley‐Liss, Inc.