A novel MSC-seeded triphasic construct for the repair of osteochondral defects.

Mesenchymal stem cells (MSC) are increasingly replacing chondrocytes in tissue engineering based research for treatment of osteochondral defects. The aim of this work was to determine whether repair of critical-size chronic osteochondral defects in an ovine model using MSC-seeded triphasic constructs would show results comparable to osteochondral autografting (OATS). Triphasic implants were engineered using a beta-tricalcium phosphate osseous phase, an intermediate activated plasma phase, and a collagen I hydrogel chondral phase. Autologous MSCs were used to seed the implants, with chondrogenic predifferentiation of the cells used in the cartilage phase. Osteochondral defects of 4.0 mm diameter were created bilaterally in ovine knees (n = 10). Six weeks later, half of the lesions were treated with OATS and half with triphasic constructs. The knees were dissected at 6 or 12 months. With the chosen study design we were not able to demonstrate significant differences between the histological scores of both groups. Subcategory analysis of O'Driscoll scores showed superior cartilage bonding in the 6-month triphasic group compared to the autograft group. The 12-month autograft group showed superior cartilage matrix morphology compared to the 12-month triphasic group. Macroscopic and biomechanical analysis showed no significant differences at 12 months. Autologous MSC-seeded triphasic implants showed comparable repair quality to osteochondral autografts in terms of histology and biomechanical testing.

Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

Impact of oxygen environment on mesenchymal stem cell expansion and chondrogenic differentiation.

INTRODUCTION: In vitro expansion and differentiation of mesenchymal stem cells (MSC) rely on specific environmental conditions, and investigations have demonstrated that one crucial factor is oxygen environment. OBJECTIVES: In order to understand the impact of oxygen tension on MSC culture and chondrogenic differentiation in vitro, we developed a mathematical model of these processes and applied it in predicting optimal assays. METHODS AND RESULTS: We compared ovine MSCs under physiologically low and atmospheric oxygen tension. Low oxygen tension improved their in vitro population growth as demonstrated by monoclonal expansion and colony forming assays. Moreover, it accelerated induction of the chondrogenic phenotype in subsequent three-dimensional differentiation cultures. We introduced a hybrid stochastic multiscale model of MSC organization in vitro. The model assumes that cell adaptation to non-physiological high oxygen tension reversibly changes the structure of MSC populations with respect to differentiation. In simulation series, we demonstrated that these changes profoundly affect chondrogenic potential of the populations. Our mathematical model provides a consistent explanation of our experimental findings. CONCLUSIONS: Our approach provides new insights into organization of MSC populations in vitro. The results suggest that MSC differentiation is largely reversible and that lineage plasticity is restricted to stem cells and early progenitors. The model predicts a significant impact of short-term low oxygen treatment on MSC differentiation and optimal chondrogenic differentiation at 10-11% pO(2).

Low oxygen expansion improves subsequent chondrogenesis of ovine bone-marrow-derived mesenchymal stem cells in collagen type I hydrogel.

BACKGROUND/OBJECTIVE: A crucial factor when investigating cartilage tissue engineering using mesenchymal stem cells (MSCs) is their application in large-animal models and preclinical trials. However, in vitro studies using cells of these model organisms must proceed. Considering that oxygen tension is an important parameter for stem cell culture, we investigated the effect of low oxygen tension during the expansion of ovine MSCs on colony-forming unit-fibroblast (CFU-F) formation, senescence and subsequent chondrogenesis in pellet culture and a collagen I hydrogel which is in clinical use for matrix-associated autologous chondrocyte transplantation (MACT). MATERIALS AND METHODS: Ovine MSCs were isolated from bone marrow aspirates and cultured at 5 and 20% O(2) in monolayer. CFU-F formation was detected by Giemsa staining. Senescence was analyzed by detection of senescence-associated beta-galactosidase and flow cytometry. Chondrogenic differentiation was carried out in pellet and collagen I hydrogel culture and assessed by gene expression, immunohistochemistry and measurement of sulfated glycosaminoglycans (sGAG). RESULTS: MSCs expanded at 5% O(2) revealed a 2-fold higher CFU-F potential and diminished senescence compared to those expanded at 20% O(2). Most notably, our results show enhanced chondrogenic differentiation in both pellet culture and the MACT-approved collagen I hydrogel. CONCLUSION: The findings demonstrate that physiologically low oxygen tension during monolayer expansion of ovine MSCs is advantageous in order to improve cartilage tissue engineering in a sheep model. The ovine system is shown to represent an appropriate basis for large-animal studies and preclinical trials on MSC-based cartilage repair.

Cartilage tissue engineering by collagen matrix associated bone marrow derived mesenchymal stem cells.

In this study we compared the in vitro formation of cartilaginous grafts composed of collagen type I hydrogel with both ovine primary articular chondrocytes (AC) and bone marrow derived mesenchymal stem cells (MSC). During 4 weeks of culture, aggregate properties were quantitatively verified, cell viability and the expression of cartilage markers were assayed. Different microscopic techniques indicated a subdivision of MSC based scaffolds into a central construct region with uniformly distributed stem cells with low levels of apoptosis, and peripheral layers of proliferative cells, which undergo differentiation. Immunohistochemical staining and quantitative measurements of sulfated glycosaminoglycans (s-GAG) of MSC hydrogels showed a significant increase in matrix deposition, mainly in outer areas. Furthermore, semi-quantitative RT-PCR of MSC specimens reflected a constant collagen type I activity with enhanced collagen type II, aggrecan and Sox9 expression which would suggest hyaline-like cartilage formation. We propose the application of MSC seeded grafts for the repair of focal cartilage lesions.