Matthias Zscharnack

Repair of Chronic Osteochondral Defects Using Predifferentiated Mesenchymal Stem Cells in an Ovine Model

Background: The use of mesenchymal stem cells (MSCs) to treat osteochondral defects caused by sports injuries or disease is of particular interest. However, there is a lack of studies in large-animal models examining the benefits of chondrogenic predifferentiation in vitro for repair of chronic osteochondral defects. Hypothesis: Chondrogenic in vitro predifferentiation of autologous MSCs embedded in a collagen I hydrogel currently in clinical trial use for matrix-associated autologous chondrocyte transplantation facilitates the regeneration of a chronic osteochondral defect in an ovine stifle joint. Study Design: Controlled laboratory study. Methods: The optimal predifferentiation period of ovine MSCs within the type I collagen hydrogel in vitro was defined by assessment of several cellular and molecular biological parameters. For the animal study, osteochondral lesions (diameter 7 mm) were created at the medial femoral condyles of the hind legs in 10 merino sheep. To achieve a chronic defect model, implantation of the ovine MSCs/hydrogel constructs was not performed until 6 weeks after defect creation. The 40 defects were divided into 4 treatment groups: (1) chondrogenically predifferentiated ovine MSC/hydrogel constructs (preMSC-gels), (2) undifferentiated ovine MSC/hydrogel constructs (unMSC-gels), (3) cell-free collagen hydrogels (CF-gels), and (4) untreated controls (UCs). Evaluation followed after 6 months. Results: With regard to proteoglycan content, cell count, gel contraction, apoptosis, compressive properties, and progress of chondrogenic differentiation, a differentiation period of 14 days in vitro was considered optimal. After 6 months in vivo, the defects treated with preMSC-gels showed significantly better histologic scores with morphologic characteristics of hyaline cartilage such as columnarization and presence of collagen type II. Conclusion: Matrix-associated autologous chondrocyte transplantation with predifferentiated MSCs may be a promising approach for repair of focal, chronic osteochondral defects. Clinical Relevance: The results suggest an encouraging method for future treatment of focal osteochondral defects to prevent progression to osteoarthritis.

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% O2 in monolayer. CFU-F formation was detected by Giemsa staining. Senescence was analyzed by detection of senescence-associated β-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% O2 revealed a 2-fold higher CFU-F potential and diminished senescence compared to those expanded at 20% O2. 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.

Matrix-Associated Implantation of Predifferentiated Mesenchymal Stem Cells Versus Articular Chondrocytes: In Vivo Results of Cartilage Repair After 1 Year

Background: The use of predifferentiated mesenchymal stem cells (MSC) leads to better histological results compared with undifferentiated MSC in sheep. This raises the need for a longer term follow-up study and comparison with a clinically established method. Hypothesis: We hypothesized that chondrogenic in vitro predifferentiation of autologous MSC embedded in a collagen I hydrogel leads to better structural repair of a chronic osteochondral defect in an ovine stifle joint after 1 year. We further hypothesized that resulting histological results would be comparable with those of chondrocyte-seeded matrix-associated autologous chondrocyte transplantation (MACT). Study Design: Controlled laboratory study. Methods: Predifferentiation period of ovine MSC within collagen gel in vitro was defined by assessment of several cellular and molecular biological parameters. For the animal study, 2 osteochondral lesions (7-mm diameter) were created at the medial femoral condyles of the hind legs in 9 sheep. Implantation of MSC gels was performed 6 weeks after defect creation. Thirty-six defects were divided into 4 treatment groups: (1) chondrogenically predifferentiated MSC gels (pre-MSC gels), (2) undifferentiated MSC gels (un-MSC gels), (3) MACT gels, and (4) untreated controls (UC). Histological, immunohistochemical, and radiological evaluations followed after 12 months. Results: After 12 months in vivo, pre-MSC gels showed significantly better histological outcome compared with un-MSC gels and UC. Compared with MACT gels, the overall scores were higher for O’Driscoll and International Cartilage Repair Society (ICRS). The repair tissue of the pre-MSC group showed immunohistochemical detection of interzonal collagen type II staining. Radiological evaluation supported superior bonding of pre-MSC gels to perilesional native cartilage. Compared with previous work by our group, no degradation of the repair tissue between 6 and 12 months in vivo, particularly in pre-MSC gels, was observed. Conclusion: Repair of chronic osteochondral defects with collagen hydrogels composed of chondrogenically predifferentiated MSC shows no signs of degradation after 1 year in vivo. In addition, pre-MSC gels lead to partially superior histological results compared with articular chondrocytes. Clinical Relevance: The results suggest an encouraging method for future treatment of focal osteochondral defects without donor site morbidity by harvesting articular chondrocytes.

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.

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.

Spatial Organization of Mesenchymal Stem Cells In Vitro—Results from a New Individual Cell-Based Model with Podia

Therapeutic application of mesenchymal stem cells (MSC) requires their extensive in vitro expansion. MSC in culture typically grow to confluence within a few weeks. They show spindle-shaped fibroblastoid morphology and align to each other in characteristic spatial patterns at high cell density. We present an individual cell-based model (IBM) that is able to quantitatively describe the spatio-temporal organization of MSC in culture. Our model substantially improves on previous models by explicitly representing cell podia and their dynamics. It employs podia-generated forces for cell movement and adjusts cell behavior in response to cell density. At the same time, it is simple enough to simulate thousands of cells with reasonable computational effort. Experimental sheep MSC cultures were monitored under standard conditions. Automated image analysis was used to determine the location and orientation of individual cells. Our simulations quantitatively reproduced the observed growth dynamics and cell-cell alignment assuming cell density-dependent proliferation, migration, and morphology. In addition to cell growth on plain substrates our model captured cell alignment on micro-structured surfaces. We propose a specific surface micro-structure that according to our simulations can substantially enlarge cell culture harvest. The ‘tool box’ of cell migratory behavior newly introduced in this study significantly enhances the bandwidth of IBM. Our approach is capable of accommodating individual cell behavior and collective cell dynamics of a variety of cell types and tissues in computational systems biology.

Isolation and Characterization of CD271⁺ Stem Cells Derived from Sheep Dermal Skin.

Background: Mesenchymal stem cells (MSCs) have great promise in the field of regenerative medicine due to their differentiation potential into several lineages. Besides the bone marrow, MSCs can be obtained from the dermis, which represents a large stem cell reservoir in the skin. Sheep provide an appropriate large animal model for preclinical studies. In this study, we focused on the isolation and characterization of MSCs from sheep dermis as an alternative to bone marrow MSCs (bmMSCs). Methods: Primary ovine cells were obtained from the dermis for comparison with bone marrow. CD271+/45- dermal MSCs (CD271-dMSCs), which were sorted by flow cytometry, and plastic-adherent bmMSCs were examined for morphology, proliferation and senescence-associated β-galactosidase activity in both low and high oxygen conditions. CD271 expression on cultured cells was assessed by flow cytometry. Adipogenic and osteogenic potentials of CD271-dMSCs were evaluated by oil red O and von Kossa staining. Chondrogenic capacity of CD271-dMSCs and CD271+/CD45- bone marrow cells (CD271-bmMSCs) was detected using immunohistochemistry and measurement of sulfated glycosaminoglycans. Results: The cell proliferation assay demonstrated no significant difference between CD271-dMSCs and bmMSCs under low oxygen conditions. Cultured CD271-dMSCs revealed much more CD271 expression compared to CD271-bmMSCs. CD271-dMSCs and CD271-bmMSCs showed basically similar expression of the cartilage-specific proteins aggrecan and collagen type II, although with a stronger staining in CD271-bmMSC-derived cultures. Remarkably, there was co-expression of CD271 and aggrecan during chondrogenic differentiation, suggesting an involvement of CD271 in chondrogenesis. Conclusion: Based on these findings, CD271-dMSCs might serve as an appropriate alternative cell source in preclinical research.

Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells-An in vitro proof-of-concept study

Due to the poor self‐healing capacities of cartilage, innovative approaches are a major clinical need. The use of in vitro expanded mesenchymal stromal cells (MSCs) in a 2‐stage approach is accompanied by cost‐, time‐, and personnel‐intensive good manufacturing practice production. A 1‐stage intraoperative procedure could overcome these drawbacks. The aim was to prove the feasibility of a point‐of‐care concept for the treatment of cartilage lesions using defined MSC subpopulations in a collagen hydrogel without prior MSC monolayer expansion. We tested 4 single marker candidates (MSCA‐1, W4A5, CD146, CD271) for their effectiveness of separating colony‐forming units of ovine MSCs via magnetic cell separation. The most promising surface marker with regard to the highest enrichment of colony‐forming cells was subsequently used to isolate a MSC subpopulation for the direct generation of a cartilage graft composed of a collagen type I hydrogel without the propagation of MSCs in monolayer. We observed that separation with CD271 sustained the highest enrichment of colony‐forming units. We then demonstrated the feasibility of generating a cartilage graft with an unsorted bone marrow mononuclear cell fraction and with a characterized CD271 positive MSC subpopulation without the need for a prior cell expansion. A reduced volume of 6.25% of the CD271 positive MSCs was needed to achieve the same results regarding chondrogenesis compared with the unseparated bone marrow mononuclear cell fraction, drastically reducing the number of nonrelevant cells. This study provides a proof‐of‐concept and reflects the potential of an intraoperative procedure for direct seeding of cartilage grafts with selected CD271 positive cells from bone marrow.

Identification of the genes GPD1 and GPD2 of Pichia jadinii

Two genes coding for proteins with a high degree of sequence similarity to glycerol-3-phosphate dehydrogenases have been isolated from the yeast Pichia jadinii. Fragments of the genes were PCR-amplified with degenerated primers from genomic DNA of P. jadinii. Clones containing the full-length genes PjGPD1 and PjGPD2 were isolated by screening genomic libraries. DNA sequencing revealed open reading frames (ORFs) of 1182 bp and 1185 bp for PjGpd1p and PjGpd2p, respectively. In a complementation study PjGPD1 rescued the growth defect of a Saccharomyces cerevisiae Δgpd1 mutant strain under osmotic stress, while complementation by PjGPD2 is temperature sensitive. The sequences of the PjGPD1 and PjGPD2 ORFs have been submitted to the EMBL Nucleotide Sequence Database under Accession No. AJ632339 and AJ632340, the sequences of the corresponding genomic DNA fragments under Accession No. AJ632341 and AJ635370, respectively.

Predifferentiated Mesenchymal Stem Cells for Osteochondral Defects: Letter

Dear Editor: We read the recent article ‘‘Repair of Chronic Osteochondral Defects Using Predifferentiated Mesenchymal Stem Cells in an Ovine Model’’ by Zscharnack et al with great interest. Clinical results of matrix-associated chondrocyte transplantation (MACT) in the treatment of chondral defects are satisfying and this technique can be seen as gold standard in the present therapy of large chondral defects. In comparison with microfracture, it offers the possibility of regeneration with hyaline-like tissue. However, advantage toward microfracture (MFX) is dearly bought by the necessity of invasive harvesting of autologous chondrocytes, leading to defect healing of donor sites and requiring an additional surgical procedure. However, recent efforts combining mesenchymal stem cells (MSCs), obtained by MFX, with artificial matrices to improve results of the MFX procedure, resulted in inferior regeneration compared with MACT. The current study directly addresses the subsequent and, in our opinion, absolutely relevant question, if in vitro predifferentiation of MSCs helps improve results. The authors attached importance to designing a study, with results that are transferable to humans, employing a chronic osteochondral defect model in large animals. Furthermore, they worked out a convincing treatment procedure that seems to be transferable to clinical application in patients. The extensive in vitro part of the work, investigating the conditions for the subsequent in vivo part, contributes markedly to the quality of the article. However, the work of the in vivo part disappointed us with regard to a few facts. All conclusions from the costly in vivo study were drawn from histologic examination. In our opinion, a biochemical, and even more a biomechanical, evaluation of the in vivo regenerate would have based the conclusions on a far more stable fundament. These additional data would characterize function of the regenerate and thereby give hints about its long-term survival. One of the most critical points in the present study is the press-fit anchoring of the gels during the surgical procedure. It is doubtable that the gels are securely prevented from dislocation during mobilization of the specimens under full weightbearing. Previous studies in comparable models have reported that a critical number of gels dislocate even when they are additionally fixed by fibrin clue or sutures. Even in clinical application of MACT, where patients avoid weightbearing for 6 weeks after surgery, delamination of the matrix is one of the major complications. Furthermore, Figure 1 of the article shows that cartilage of the sheep’s condyle is rather thin compared with that of humans. This even more brings into question press-fit stability under full weightbearing. Also, if the authors have shown successful survival of implants in situ for 6 weeks in single sheep employing quantum dot-labeling (Figure 6), toluidine staining in Figure 8 of the article supports the assumption that some gels were dislocated after the surgical procedure. Here, the worst results of each treatment group show no residual matrix, resulting in persistence of full cartilage defects. This finding surely contributes to the general high standard deviation in the present study. On the other hand, the best results shown in Figure 8, with full defect regeneration in all groups, including untreated controls, make it questionable if the 7-mm diameter represents a critical-size defect. Here, the result of the predifferentiated MSC-seeded gel group appears as good as native cartilage, raising the question if the histological slide has missed the regenerate. Another critical limitation of the study design is creation of 2 randomized treated defects in 1 knee. Application of 2 different treatments in 1 joint cavity creates danger of their interaction. Although the authors state that they are conscious of that danger and quote that no obvious influence between the different defects was observed, it is well known that hydrogels loaded with cells release cytokines and cells to their environment and therewith to the joint cavity. Here, it is likely that stem cells, either differentiated or undifferentiated, released from gels into the joint critically interacted with healing of defects, either treated with unloaded gels or even if left empty. On the other hand, anabolic cytokines released from MSCs may have improved healing in defects treated by other methods in the same compartment. Additionally, chondrogenic differentiated MSCs incorporated to matrices may have paracrinely influenced undifferentiated MSCs applied to the same joint cavity. Thus, it is likely that interactions of the different treatment methods have contributed to the high standard deviation observed within the groups. Regarding these facts, we are wondering why the authors have not randomized the knees (instead of randomizing the single defects), meaning same treatment in both defects of 1 knee and thereby avoiding uncontrolled interaction. An additional factor surely influencing the different treatment regimens is the depth of the set defect. How did the authors confirm, that each defect was 2 mm in depth? Did the drill have a mechanical stop? The authors note that the subchondral bone layer was not opened during drilling the procedure to avoid migration of MSCs to the defect. Nonetheless, in Figure 1 the subchondral layer seems to be affected, which is in line with hypertrophy of subchondral bone in several treatments represented in Figure 8. This bone hypertrophy is frequently occurring The American Journal of Sports Medicine, Vol. 39, No. 6 DOI: 10.1177/0363546511410382 2011 The Author(s)