An ex vivo human cartilage repair model to evaluate the potency of a cartilage cell transplant

Bartz, Christoph; Meixner, Miriam; Giesemann, P.; Roël, Giulietta; Bulwin, Grit-Carsta; Smink, Jeske J.

doi:10.1186/s12967-016-1065-8

Cited by 38 publications

(41 citation statements)

References 27 publications

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“…Only 12-26 weeks after implantation of the single cell suspension, cartilage matrix develops (Becher et al, 2017;Krill et al, 2018). The major advantage of using spheroids instead of single cells is that, because of the high cell-cell contacts and cell density in 3D preculture, they already have developed a tissue specific ECM, before they are used for implantation (Bartz et al, 2016;De Moor et al, 2019). We believe the use of spheroids will lead to a less fibrous tissue than traditional ACI because they already possess a GAG-and collagen II-rich ECM.…”

Section: Discussionmentioning

confidence: 99%

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids

Moor

Fernandez

Vercruysse

et al. 2020

Front. Bioeng. Biotechnol.

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To date, the treatment of articular cartilage lesions remains challenging. A promising strategy for the development of new regenerative therapies is hybrid bioprinting, combining the principles of developmental biology, biomaterial science, and 3D bioprinting. In this approach, scaffold-free cartilage microtissues with small diameters are used as building blocks, combined with a photo-crosslinkable hydrogel and subsequently bioprinted. Spheroids of human bone marrow-derived mesenchymal stem cells (hBM-MSC) are created using a high-throughput microwell system and chondrogenic differentiation is induced during 42 days by applying chondrogenic culture medium and low oxygen tension (5%). Stable and homogeneous cartilage spheroids with a mean diameter of 116 ± 2.80 µm, which is compatible with bioprinting, were created after 14 days of culture and a glycosaminoglycans (GAG)and collagen II-positive extracellular matrix (ECM) was observed. Spheroids were able to assemble at random into a macrotissue, driven by developmental biology tissue fusion processes, and after 72 h of culture, a compact macrotissue was formed. In a directed assembly approach, spheroids were assembled with high spatial control using the bio-ink based extrusion bioprinting approach. Therefore, 14-day spheroids were combined with a photo-crosslinkable methacrylamide-modified gelatin (gelMA) as viscous printing medium to ensure shape fidelity of the printed construct. The photo-initiators Irgacure 2959 and Li-TPO-L were evaluated by assessing their effect on bio-ink properties and the chondrogenic phenotype. The encapsulation in gelMA resulted in further chondrogenic maturation observed by an increased production of GAG and a reduction of collagen I. Moreover, the use of Li-TPO-L lead to constructs with lower stiffness which induced a decrease of collagen I and an increase in GAG and collagen II production. After 3D bioprinting, spheroids remained viable and the cartilage phenotype was maintained. Our findings demonstrate that hBM-MSC spheroids are able to differentiate into cartilage microtissues and display a geometry compatible with 3D bioprinting. Furthermore, for hybrid bioprinting of these spheroids,

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Section: Discussionmentioning

confidence: 99%

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids

Moor

Fernandez

Vercruysse

et al. 2020

Front. Bioeng. Biotechnol.

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“…These results argue for an advantage of cell-loaded versus cell-free collagen implants, in agreement with earlier studies on cellseeded or cell-containing implants based on collagen or other cartilage replacement materials. [41][42][43][44] Alternatively, in vivo microfracturing of the subchondral bone plate below the cartilage implant can be used to favor cell immigration into initially cell-free implants and optimize the conditions for successful cartilage repair. 39,40,45 Cell-free and cell-loaded collagen implants both showed progressive deposition of the matrix molecule aggrecan and a stable protein content of collagen 2 throughout culture, again demonstrating the cytocompatibility of the implant material and the active production of cartilage-specific matrix molecules by vital chondrocytes.…”

Section: Discussionmentioning

confidence: 99%

In VitroAnalysis of Cartilage Regeneration Using a Collagen Type I Hydrogel (CaReS) in the Bovine Cartilage Punch Model

et al. 2018

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Objective Limitations of matrix-assisted autologous chondrocyte implantation to regenerate functional hyaline cartilage demand a better understanding of the underlying cellular/molecular processes. Thus, the regenerative capacity of a clinically approved hydrogel collagen type I implant was tested in a standardized bovine cartilage punch model. Methods Cartilage rings (outer diameter 6 mm; inner defect diameter 2 mm) were prepared from the bovine trochlear groove. Collagen implants (± bovine chondrocytes) were placed inside the cartilage rings and cultured up to 12 weeks. Cartilage-implant constructs were analyzed by histology (hematoxylin/eosin; safranin O), immunohistology (aggrecan, collagens 1 and 2), and for protein content, RNA expression, and implant push-out force. Results Cartilage-implant constructs revealed vital morphology, preserved matrix integrity throughout culture, progressive, but slight proteoglycan loss from the "host" cartilage or its surface and decreasing proteoglycan release into the culture supernatant. In contrast, collagen 2 and 1 content of cartilage and cartilage-implant interface was approximately constant over time. Cell-free and cell-loaded implants showed (1) cell migration onto/into the implant, (2) progressive deposition of aggrecan and constant levels of collagens 1 and 2, (3) progressively increased mRNA levels for aggrecan and collagen 2, and (4) significantly augmented push-out forces over time. Cell-loaded implants displayed a significantly earlier and more long-lasting deposition of aggrecan, as well as tendentially higher push-out forces. Conclusion Preserved tissue integrity and progressively increasing cartilage differentiation and push-out forces for up to 12 weeks of cultivation suggest initial cartilage regeneration and lateral bonding of the implant in this in vitro model for cartilage replacement materials.

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“…GAG secretion was analysed with standard 1,9-dimethylmethylene blue assay using chondroitin sulphate sodium salt from shark (Sigma-Aldrich) to generate standard curves as described before [ 19 ]. Standard and samples of each donor were measured in triplicates.…”

Section: Methodsmentioning

confidence: 99%

Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells

Schubert¹,

Smink²,

Pumberger³

et al. 2018

J Orthop Surg Res

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BackgroundThe lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell’s behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture.MethodsHuman AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco’s Modified Eagle’s Medium (DMEM) or Ham’s F-12 medium (Ham’s F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation.ResultsAF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham’s F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham’s F-12.ConclusionThe impact of the different media itself on disc cell’s behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

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An ex vivo human cartilage repair model to evaluate the potency of a cartilage cell transplant

Cited by 38 publications

References 27 publications

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids

Hybrid Bioprinting of Chondrogenically Induced Human Mesenchymal Stem Cell Spheroids

In VitroAnalysis of Cartilage Regeneration Using a Collagen Type I Hydrogel (CaReS) in the Bovine Cartilage Punch Model

Standardisation of basal medium for reproducible culture of human annulus fibrosus and nucleus pulposus cells

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