Strategies to overcome the limited availability of human articular chondrocytes and their tendency to dedifferentiate during expansion are required to advance their clinical use and to engineer functional cartilage on par with native articular cartilage. This work sought to determine whether a biochemical factor (transforming growth factor-β1 [T]), a biophysical agent (chondroitinase-ABC [C]), and a collagen crosslinking enzyme (lysyl oxidase-like 2 [L]) are efficacious in forming three-dimensional human neocartilage from expanded human articular chondrocytes. Among the treatment regimens, the combination of the three stimuli (TCL treatment) led to the most robust glycosaminoglycan content, total collagen content, and type II collagen production. In particular, TCL treatment synergistically increased tensile stiffness and strength of human neocartilage by 3.5-fold and 3-fold, respectively, over controls. Applied to two additional donors, the beneficial effects of TCL treatment appear to be donor independent; tensile stiffness and strength were increased by up to 8.5-fold and 3-fold, respectively, over controls. The maturation of human neocartilage in response to TCL treatment was examined following 5 and 8 weeks of culture, demonstrating maintenance or further enhancement of functional properties. The present study identifies a novel strategy for engineering human articular cartilage using serially passaged chondrocytes.

Effective early disease modifying options for osteoarthritis remain lacking. Tissue engineering approach to generate cartilage in vitro has emerged as a promising option for articular cartilage repair and regeneration. Signaling molecules and matrix modifying agents, derived from knowledge of cartilage development and homeostasis, have been used as biochemical stimuli toward cartilage tissue engineering and have led to improvements in the functionality of engineered cartilage. Clinical translation of neocartilage faces challenges, such as phenotypic instability of the engineered cartilage, poor integration, inflammation, and catabolic factors in the arthritic environment; these can all contribute to failure of implanted neocartilage. A comprehensive understanding of signaling molecules involved in osteoarthritis pathogenesis and their actions on engineered cartilage will be crucial. Thus, while it is important to continue deriving inspiration from cartilage development and homeostasis, it has become increasingly necessary to incorporate knowledge from osteoarthritis pathogenesis into cartilage tissue engineering.

OBJECTIVE: The medial femoral condyle of the knee exhibits some of the highest incidences of chondral degeneration. However, a dearth of healthy human tissues has rendered it difficult to ascertain whether cartilage in this compartment possesses properties that predispose it to injuries. Assessment of young, healthy tissue would be most representative of the tissues intrinsic properties. DESIGN: This work examined the topographical differences in tribological, tensile, and compressive properties of young (n = 5, 26.2 ± 5.6 years old), healthy, human medial femoral condyles, obtained from viable allograft specimens. Corresponding to clinical incidences of pathology, it was hypothesized that the lowest mechanical properties would be found in the posterior region of the medial condyle, and that tissue composition would correspond to the established structure-function relationships of cartilage. RESULTS: Youngs modulus, ultimate tensile strength, aggregate modulus, and shear modulus in the posterior region were 1.0-, 2.8-, 1.1-, and 1.0-fold less than the values in the anterior region, respectively. Surprisingly, although glycosaminoglycan content is thought to correlate with compressive properties, in this study, the aggregate and shear moduli correlated more robustly to the amount of pyridinoline crosslinks per collagen. Also, the coefficient of friction was anisotropic and ranged 0.22-0.26 throughout the condyle. CONCLUSION: This work showed that the posteromedial condyle displays lower tensile and compressive properties, which correlate to collagen crosslinks and may play a role in this regions predisposition to injuries. Furthermore, new structure-function relationships may need to be developed to account for the role of collagen crosslinks in compressive properties.

To gain regulatory approval for the clinical use of knee biologics and devices in humans, translational large-animal studies are typically required. Animal models that permit second-look arthroscopy are valuable because they allow for longitudinal assessment of the treated tissue without needing to sacrifice the animal. The minipig is an ideal preclinical animal model for the investigation of therapies for the knee, in part because arthroscopy can be performed in its stifle (knee) joint with the use of standard surgical equipment used in humans. The purpose of this Technical Note is to describe a reproducible technique for diagnostic arthroscopy of the minipig stifle (knee) joint.

Background

Although the toxic effects of bupivacaine on chondrocyte monolayer culture have been well described, its cellular and mechanical effects on native and engineered articular cartilage remain unclear. For the repair of articular cartilage defects, fresh autologous and allogenic cartilage grafts are commonly used, and engineered cell-based therapies are emerging. The outcome of grafting therapies aimed at repairing damaged cartilage relies largely on maintaining proper viability and mechanical suitability of the donor tissues.

Purpose

To investigate the in vitro effects of single bupivacaine exposure on the viability and mechanics of 2 cartilage graft types: native articular cartilage and engineered neocartilage.

Study design

Controlled laboratory study.

Methods

Articular cartilage explants were harvested from the bovine stifle femoral condyles, and neocartilage constructs were engineered from bovine stifle chondrocytes using the self-assembling process, a scaffold-free approach to engineer cartilage tissue. Both explants and neocartilage were exposed to chondrogenic medium containing a clinically applicable bolus of 0.5%, 0.25%, or 0% (control) bupivacaine for 1 hour, followed by fresh medium wash and exchange. Cell viability and matrix content (collagen and glycosaminoglycan) were assessed at t = 24 hours after treatment, and compressive mechanical properties were assessed with creep indentation testing at t = 5 to 6 days after treatment.

Results

Single bupivacaine exposure was chondrotoxic in both explants and neocartilage, with 0.5% bupivacaine causing a significant decrease in chondrocyte viability compared with the control condition (55.0% ± 13.4% vs 71.9% ± 13.5%; P < .001). Bupivacaine had no significant effect on matrix content for either tissue type. There was significant weakening of the mechanical properties in the neocartilage when treated with 0.5% bupivacaine compared with control, with decreased aggregate modulus (415.8 ± 155.1 vs 660.3 ± 145.8 kPa; P = .003), decreased shear modulus (143.2 ± 14.0 vs 266.5 ± 89.2 kPa; P = .002), and increased permeability (14.7 ± 8.1 vs 6.6 ± 1.7 × 10^-15 m⁴/Ns; P = .009). Bupivacaine exposure did not have a significant effect on the mechanical properties of native cartilage explants.

Conclusion

Single bupivacaine exposure resulted in significant chondrotoxicity in native explants and neocartilage and significant weakening of mechanical properties of neocartilage. The presence of abundant extracellular matrix does not appear to confer any additional resistance to the toxic effects of bupivacaine.

Clinical relevance

Clinicians should be judicious regarding the use of intra-articular bupivacaine in the setting of articular cartilage repair.

Human articular chondrocytes (hACs) are scarce and lose their chondrogenic potential during monolayer passaging, impeding their therapeutic use. This study investigated (a) the translatability of conservative chondrogenic passaging and aggregate rejuvenation on restoring chondrogenic properties of hACs passaged up to P9; and (b) the efficacy of a combined treatment of transforming growth factor-beta 1 (TGF-β1) (T), chondroitinase-ABC (C), and lysyl oxidase-like 2 (L), collectively termed TCL, on engineering functional human neocartilage via the self-assembling process, as a function of passage number up to P11. Here, we show that aggregate rejuvenation enhanced glycosaminoglycan (GAG) content and type II collagen staining at all passages and yielded human neocartilage with chondrogenic phenotype present up to P7. Addition of TCL extended the chondrogenic phenotype to P11 and significantly enhanced GAG content and type II collagen staining at all passages. Human neocartilage derived from high passages, treated with TCL, displayed mechanical properties that were on par with or greater than those derived from low passages. Conservative chondrogenic passaging and aggregate rejuvenation may be a viable new strategy (a) to address the perennial problem of chondrocyte scarcity and (b) to successfully rejuvenate the chondrogenic phenotype of extensively passaged cells (up to P11). Furthermore, tissue engineering human neocartilage via self-assembly in conjunction with TCL treatment advances the clinical use of extensively passaged human chondrocytes for cartilage repair.

Injuries to articular cartilage and menisci can lead to cartilage degeneration that ultimately results in arthritis. Different forms of arthritis affect ~50 million people in the USA alone, and it is therefore crucial to identify methods that will halt or slow the progression to arthritis, starting with the initiating events of cartilage and meniscus defects. The surgical approaches in current use have a limited capacity for tissue regeneration and yield only short-term relief of symptoms. Tissue engineering approaches are emerging as alternatives to current surgical methods for cartilage and meniscus repair. Several cell-based and tissue-engineered products are currently in clinical trials for cartilage lesions and meniscal tears, opening new avenues for cartilage and meniscus regeneration. This Review provides a summary of surgical techniques, including tissue-engineered products, that are currently in clinical use, as well as a discussion of state-of-the-art tissue engineering strategies and technologies that are being developed for use in articular cartilage and meniscus repair and regeneration. The obstacles to clinical translation of these strategies are also included to inform the development of innovative tissue engineering approaches.

Dermis-isolated adult stem (DIAS) cells, abundantly available, are attractive for regenerative medicine. Strategies have been devised to isolate and to chondroinduce DIAS cells from various animals. This study aimed to characterize DIAS cells from human abdominal skin (human dermis-isolated adult stem [hDIAS] cells) and to compare and to refine various chondroinduction regimens to form functional neocartilage constructs. The stemness of hDIAS cells was verified (Phase I), three chondroinduction pretreatments were compared (Phase II), and, from these, one regimen was carried forward for refinement in Phase III for improving the mechanical properties of hDIAS cell-derived constructs. Multilineage differentiation and mesenchymal stem cell markers were observed. Among various chondroinduction pretreatments, the nodule formation pretreatment yielded constructs at least 72% larger in diameter, with higher glycosaminoglycan (GAG) content by 44%, compared with other pretreatments. Furthermore, it was found that culturing cells on nontissue culture-treated surfaces yielded constructs (1) on par with constructs derived from aggrecan-coated surfaces and (2) with superior mechanical properties than constructs derived from cells cultured on tissue culture-treated surfaces. After the nodule formation pretreatment, combined supplementation of TGF-β1, IGF-I, and fetal bovine serum significantly enhanced aggregate modulus and shear modulus by 75% and 69%, respectively, over the supplementation by TGF-β1 alone. In summary, human skin-derived DIAS cells are responsive to chondroinduction for forming neocartilage. Furthermore, the mechanical properties of the resultant human constructs can be improved by treatments shown to be efficacious in animal models. Advances made toward tissue-engineering cartilage using animal cells were shown to be applicable to hDIAS cells for cartilage repair and regeneration.

Abundance and accessibility render skin-derived stem cells an attractive cell source for tissue engineering applications. Toward assessing their utility, the variability of constructs engineered from human dermis-isolated adult stem (hDIAS) cells was examined with respect to different anatomical locations (foreskin, breast, and abdominal skin), both in vitro and in a subcutaneous, athymic mouse model. All anatomical locations yielded hDIAS cells with multi-lineage differentiation potentials, though adipogenesis was not seen for foreskin-derived hDIAS cells. Using engineered cartilage as a model, tissue engineered constructs from hDIAS cells were compared. Construct morphology differed by location. The mechanical properties of human foreskin- and abdominal skin-derived constructs were similar at implantation, remaining comparable after 4 additional weeks of culture in vivo. Breast skin-derived constructs were not mechanically testable. For all groups, no signs of abnormality were observed in the host. Addition of aggregate redifferentiation culture prior to construct formation improved chondrogenic differentiation of foreskin-derived hDIAS cells, as evident by increases in glycosaminoglycan and collagen contents. More robust Alcian blue staining and homogeneous cell populations were also observed compared to controls. Human DIAS cells elicited no adverse host responses, reacted positively to chondrogenic regimens, and possessed multi-lineage differentiation potential with the caveat that efficacy may differ by anatomical origin of the skin. Taken together, these results suggest that hDIAS cells hold promise as a potential cell source for a number of tissue engineering applications.