Effect of Press-Fit Size on Insertion Mechanics and Cartilage Viability in Human and Ovine Osteochondral Grafts

AUTHORS

R.P. Suderman, M.B. Hurtig, M.D. Grynpas, P.R.T. Kuzyk, and A. Changoor

ABSTRACT

Objective

The osteochondral allograft procedure uses grafts constructed larger than the recipient site to stabilize the graft, in what is known as the press-fit technique. This research aims to characterize the relationships between press-fit size, insertion forces, and cell viability in ovine and human osteochondral tissue.

Design

Human (4 donors) and ovine (5 animals) articular joints were used to harvest osteochondral grafts (4.55 mm diameter, N = 33 Human, N = 35 Ovine) and create recipient sites with grafts constructed to achieve varying degrees of press fit (0.025-0.240 mm). Donor grafts were inserted into recipient sites while insertion forces were measured followed by quantification of chondrocyte viability and histological staining to evaluate the extracellular matrix.

Results

Both human and ovine tissues exhibited similar mechanical and cellular responses to changes in press-fit. Insertion forces (Human: 3-169 MPa, Ovine: 36-314 MPa) and cell viability (Human: 16%-89% live, Ovine: 2%-76% live) were correlated to press-fit size for both human (force: r = 0.539, viability: r = −0.729) and ovine (force: r = 0.655, viability: r = −0.714) tissues. In both species, a press-fit above 0.14 mm resulted in reduced cell viability below a level acceptable for transplantation, increased insertion forces, and reduced linear correlation to press-fit size compared to samples with a press-fit below 0.14 mm.

Conclusions

Increasing press-fit size required increased insertion forces and resulted in reduced cell viability. Ovine and human osteochondral tissues responded similarly to impact insertion and varying press-fit size, providing evidence for the use of the ovine model in allograft-related research.

Piezo1 expression in chondrocytes controls endochondral ossification and osteoarthritis development

AUTHORS

Laura J. Brylka, Assil-Ramin Alimy, Miriam E. A. Tschaffon-Müller, Shan Jiang, Tobias Malte Ballhause, Anke Baranowsky, Simon von Kroge, Julian Delsmann, Eva Pawlus, Kian Eghbalian, Klaus Püschel, Astrid Schoppa, Melanie Haffner-Luntzer, David J. Beech, Frank Timo Beil, Michael Amling, Johannes Keller, Anita Ignatius, Timur A. Yorgan, Tim Rolvien & Thorsten Schinke

ABSTRACT

Piezo proteins are mechanically activated ion channels, which are required for mechanosensing functions in a variety of cell types. While we and others have previously demonstrated that the expression of Piezo1 in osteoblast lineage cells is essential for bone-anabolic processes, there was only suggestive evidence indicating a role of Piezo1 and/or Piezo2 in cartilage. Here we addressed the question if and how chondrocyte expression of the mechanosensitive proteins Piezo1 or Piezo2 controls physiological endochondral ossification and pathological osteoarthritis (OA) development. Mice with chondrocyte-specific inactivation of Piezo1 (Piezo1Col2a1Cre), but not of Piezo2, developed a near absence of trabecular bone below the chondrogenic growth plate postnatally. Moreover, all Piezo1Col2a1Cre animals displayed multiple fractures of rib bones at 7 days of age, which were located close to the growth plates. While skeletal growth was only mildly affected in these mice, OA pathologies were markedly less pronounced compared to littermate controls at 60 weeks of age. Likewise, when OA was induced by anterior cruciate ligament transection, only the chondrocyte inactivation of Piezo1, not of Piezo2, resulted in attenuated articular cartilage degeneration. Importantly, osteophyte formation and maturation were also reduced in Piezo1Col2a1Cre mice. We further observed increased Piezo1 protein abundance in cartilaginous zones of human osteophytes. Finally, we identified Ptgs2 and Ccn2 as potentially relevant Piezo1 downstream genes in chondrocytes. Collectively, our data do not only demonstrate that Piezo1 is a critical regulator of physiological and pathological endochondral ossification processes, but also suggest that Piezo1 antagonists may be established as a novel approach to limit osteophyte formation in OA.

High-glutathione mesenchymal stem cells isolated using the FreSHtracer probe enhance cartilage regeneration in a rabbit chondral defect model

AUTHORS

Gun Hee Cho, Hyun Cheol Bae, Won Young Cho, Eui Man Jeong, Hee Jung Park, Ha Ru Yang, Sun Young Wang, You Jung Kim, Dong Myung Shin, Hyung Min Chung, In Gyu Kim & Hyuk-Soo Han

ABSTRACT

Background

Mesenchymal stem cells (MSCs) are a promising cell source for cartilage regeneration. However, the function of MSC can vary according to cell culture conditions, donor age, and heterogeneity of the MSC population, resulting in unregulated MSC quality control. To overcome these limitations, we previously developed a fluorescent real-time thiol tracer (FreSHtracer) that monitors cellular levels of glutathione (GSH), which are known to be closely associated with stem cell function. In this study, we investigated whether using FreSHtracer could selectively separate high-functioning MSCs based on GSH levels and evaluated the chondrogenic potential of MSCs with high GSH levels to repair cartilage defects in vivo.

Methods

Flow cytometry was conducted on FreSHtracer-loaded MSCs to select cells according to their GSH levels. To determine the function of FreSHtracer-isolated MSCs, mRNA expression, migration, and CFU assays were conducted. The MSCs underwent chondrogenic differentiation, followed by analysis of chondrogenic-related gene expression. For in vivo assessment, MSCs with different cellular GSH levels or cell culture densities were injected in a rabbit chondral defect model, followed by histological analysis of cartilage-regenerated defect sites.

Results

FreSHtracer successfully isolated MSCs according to GSH levels. MSCs with high cellular GSH levels showed enhanced MSC function, including stem cell marker mRNA expression, migration, CFU, and oxidant resistance. Regardless of the stem cell tissue source, FreSHtracer selectively isolated MSCs with high GSH levels and high functionality. The in vitro chondrogenic potential was the highest in pellets generated by MSCs with high GSH levels, with increased ECM formation and chondrogenic marker expression. Furthermore, the MSCs’ function was dependent on cell culture conditions, with relatively higher cell culture densities resulting in higher GSH levels. In vivo, improved cartilage repair was achieved by articular injection of MSCs with high levels of cellular GSH and MSCs cultured under high-density conditions, as confirmed by Collagen type 2 IHC, Safranin-O staining and O’Driscoll scores showing that more hyaline cartilage was formed on the defects.

Conclusion

FreSHtracer selectively isolates highly functional MSCs that have enhanced in vitro chondrogenesis and in vivo hyaline cartilage regeneration, which can ultimately overcome the current limitations of MSC therapy.

Spaceflight and hind limb unloading induces an arthritic phenotype in knee articular cartilage and menisci of rodents

AUTHORS

Andy T. Kwok, Nequesha S. Mohamed, Johannes F. Plate, Raghunatha R. Yammani, Samuel Rosas, Ted A. Bateman, Eric Livingston, Joseph E. Moore, Bethany A. Kerr, Jingyun Lee, Cristina M. Furdui, Li Tan, Mary L. Bouxsein, Virginia L. Ferguson, Louis S. Stodieck, David C. Zawieja, Michael D. Delp, Xiao W. Mao & Jeffrey S. Willey

ABSTRACT

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.