Regenerative treatment of focal hyaline cartilage defects could prevent or delay the development of secondary osteoarthritis. Current surgical techniques result partly in i) the formation of mechanically inferior fibrous cartilage or ii) present the disadvantage of the donor site morbidity from harvesting cartilage biopsy as well as iii) the dedifferentiation of chondrocytes due to in vitro expansion and iv) the reduced re-differentiation potential of in vitro expanded chondrocytes.
The self-healing capacities of injured and degenerated articular cartilage revealed a promising target cell population for a regenerative, autologous treatment of these defects using mesenchymal stromal cells (MSCs). Several case studies, randomized and controlled clinical studies showed the general ability of autologous, bone marrow-derived, expanded MSC transplantation to regenerate articular cartilage lesions [1]. However, these two-stage approaches are based on time- and cost-consuming expansion of MSCs under good manufacturing practice (GMP) conditions and hold a risk of contamination during this process.
In 2010, CD271, the low-affinity nerve growth factor receptor, was described as a suitable surface marker to enrich MSCs from human bone marrow aspirate intraoperatively [2].
The aim of the present dissertation was to investigate the feasibility of generating cartilage grafts from either ovine (study no. 1) and human (study no. 2) non-expanded CD271+ bone marrow cells in a collagen type I hydrogel.
Study no. 1 (“Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells - An in vitro proof-of-concept study”) investigated several surface marker candidates for the prospective MSC separation and examined their potential of resulting colony-forming units, respective their yield of potent MSCs [3]. This study was conducted with ovine bone marrow samples. CD271 was the most effective surface marker to isolate the target cell population. Subsequently, CD271+, CD271- and unseparated mononuclear cells (MNCs), containing the MSCs, were used to generate cartilage grafts without an expansion of these cells in monolayer culture. It could be proven, that ovine CD271+ cells were able to generate a potent hyaline cartilage graft.
Study no. 2 (“Single-stage preparation of human cartilage grafts generated from bone marrow-derived CD271+ mononuclear cells”) was performed as the final translational step from animal-derived bone marrow to human donor material and is therefore strengthening the therapeutically focus of the entire work [4].
Briefly, eight bone marrow aspirates were used for MNC isolation and subsequent magnetic cell separation (MACS). The resulting CD271+ and CD271- MNCs were compared to unseparated MNCs. Subsequently, they were seeded in a clinically approved collagen type I hydrogel and cultivated for up to 5 weeks to investigate the progression of the chondrogenic differentiation processes. Graft analysis included cell viability visualization by live/dead staining, determination of the DNA and the secreted sulphated glycosaminoglycan (sGAG) content as well as the immunohistochemical staining for typical chondrogenic differentiation markers and the extracellular matrix molecules aggrecan and collagen type II.
A proliferation of cells in the generated grafts was shown of CD271+ and unsep, but not CD271- MNCs. Hence, the cell number was 2.8-fold higher after 35 days compared to the first day for CD271+ MNCs grafts, while CD271- MNCs did not proliferate in the grafts and unsep MNCs showed only a slight increase in cell number.
The chondrogenic potential was measured by quantification of freshly produced sGAGs and the expression of chondrogenic markers. In grafts with CD271+ MNCs, sGAG production increased over time and reached its maximum at day 35, whereas grafts with CD271- MNCs showed no measurable sGAG deposition. The amount of sGAG in unsep MNC grafts increased only slightly over the whole cultivation period. Aggrecan and collagen type II staining varied considerably between the MNCs donors. Collagen type II positive staining was observed in CD271+ MNC grafts (5/8 donors) and unsep MNC (2/8) grafts. In comparison to macroscopically healthy cartilage, three-dimensional grafts of the CD271+ group yielded a proceeding extracellular matrix production.
In summary, CD271+ MNCs showed the highest proliferation rate, cell viability, sGAG deposition and cartilage marker expression compared to the CD271- or unseparated MNC fractions in in vitro generated three-dimensional cartilage grafts.
Therefore, the presented work demonstrated the feasibility of generating a cartilage graft from CD271+ bone marrow-derived MNCs in a clinically approved collagen type I hydrogel without a previous monolayer expansion of these cells. This will enable the intraoperative purification of CD271+ MNCs, which contain the majority of colony-forming MSCs, by MACS technology. The clinical application will be possible with currently available and clinical approved cell separation devices.
Providing a cartilage graft with non-expanded CD271+ MNCs by a fast and simple intraoperative therapeutic approach fulfils the need for a “single-step, tissue-engineered solution to focal cartilage defects, and elimination of the morbidity of the donor defect” as requested by the editors of the journal Arthroscopy [5].
References of the summary
1. Filardo G, et al. (2016). Stem cells in articular cartilage regeneration. J Orthop Surg Res 11:42.
2. Jones E, et al. (2010). Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells. Arthritis Rheum. 62:1944–1954.
3. Petters O, et al. (2018). Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells-An in vitro proof-of-concept study. J Tissue Eng Regen Med.
4. Petters O, et al. (2018). Single-Stage Preparation of Human Cartilage Grafts Generated from Bone Marrow-Derived CD271+ Mononuclear Cells. Stem Cells Dev 27:545–555.
5. Lubowitz JH and GG Poehling. (2009). Saving our cells: Advances in tissue engineering for focal cartilage defects. Arthroscopy: the journal of arthroscopic & related surgery: official publication of the Arthroscopy Association of North America and the International Arthroscopy Association 25:115–116.:1 Introduction 1
1.1 Articular cartilage 1
1.2 Cartilage lesions 2
1.3 Self-healing capability of articular cartilage 3
1.4 Treatment option for cartilage lesions 4
1.5 Mesenchymal stromal cells in cartilage regeneration 6
2 Rationale 8
3 Publication manuscripts 9
Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells - An in vitro proof-of-concept study 9
Single-stage preparation of human cartilage grafts generated from bone marrow-derived CD271+ mononuclear cells 21
4 Summary 33
5 References 35
6 Appendix 42
7 Declaration of Authorship 47
8 Curriculum vitae 48
9 Publications 49
10 Acknowledgements 49
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:33679 |
| Date | 01 April 2019 |
| Creators | Petters, Oliver |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/acceptedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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