Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh‐viscosity alginate

Abstract

One major problem of current cartilage repair techniques is that three-dimensional encapsulated mesenchymal progenitor cells frequently differentiate into hypertrophic cells that express type X collagen and osteogenic marker genes. Studies on wild-type cells of murine mesenchymal C3H10T1/2 progenitor cells as well as on cells transfected with cDNA encoding for bone morphogenetic protein (BMP)-2 or -4 in alginate revealed that the formation o f markers for osteogenesis and chondrogenic hypertrophy apparently depended on the BMP-transfection. Cells were encapsulated in ultrahigh-viscosity, clinical grade alginate and differentiation was studied over a period of 17 days. Consistent with results published previously (Biomaterials, 2002;23:2003) staining with haematoxylin--eosin or Alcian blue. immunohistochemical analysis, and quantitative RT-PCR confirmed the expression of chondrogenic markers (chondroitin-4-and -6-sulfate as well as type I1 collagen). Production of chondrogenic markers was particularly high in BMP-4 transfected cells. Hypertrophic chondrogenesis did not occur in BMP-4 transfected cells, as revealed by measurement of type X collagen, but could be demonstrated for wild-type cells and to some extent for BMP-2 transfected cells. The osteogenic markers, type 1 collagen, alkaline phosphatase, and Cbfal were upregulated in all cell lines even though the levels and the time of upregulation differed significantly. In any case, the markers were less and only very shortly expressed in BMP-4 transfected cells as revealed quantitatively by real time RT-PCR. Thus, the in vitro results suggested that BMP-4 is a very promising candidate for suppressing chondrogenic hypertrophy, while simultaneously enhancing the production of chondrogenic components.

Differentiation of human mesenchymal stem cells and articular chondrocytes: Analysis of chondrogenic potential and expression pattern of differentiation‐related transcription factors

“…The high collagen type X expression is in accordance with results obtained from murine MSCs. 13 We further observed that the age of the donors correlated to their ability to produce matrix, with a high matrix production in the younger donors. MSCs A produced almost no cartilage-like matrix compared to articular chondrocytes and MSCs B .…”

Cartilage repair using bone morphogenetic protein 4 and muscle‐derived stem cells

“…Studies involving embryonic stem cells and mesenchymal progenitor cells also have demonstrated the beneficial effect of BMP-4 on chondrogenic differentiation in vitro (30,31,46). Other research groups have used the culturing of cells in a micromass pellet culture system as an assay for chondrogenesis, and this approach appears to be optimal for assessing the chondrogenic differentiation of mesenchymal stem cells (35,(41)(42)(43).…”

“…Many researchers have chosen to focus on the study of cellseeded matrices in an effort to engineer synthetic cartilage in vitro for implantation in vivo. The use of cells suspended in fibrin glue instead of solid grafts enables the suspended cells to settle even in narrow clefts, and fast-forming clots are able to prevent any initial displacement (31). Research has demonstrated that fibrin glue has no adverse effects on cell viability 440 KURODA ET AL and is a suitable matrix for applications designed to promote chondrogenesis (49).…”

“…Studies performed in vitro and in the developing skeleton have led to the identification of BMP-4 as a promising candidate for the promotion of chondrogenesis (29,30). Steinert et al observed particularly high expression of chondrogenic markers in populations of BMP-4-transfected mesenchymal progenitor cells (31).…”

A new validation method for clinical grade micro-encapsulation: quantitative high speed video analysis of alginate capsule

“…Furthermore cells behave in a more natural way, retaining more physiological functionality, if they have three dimensional environments. Chondrocytes, for example, produce cartilage typical collagen II only when they are cultured in three dimensional environments (Steinert et al 2003). The increasing interest of biomedicine in cell-matrix constructs promotes the development of high throughput culture techniques (Tendulkar et al 2012) and increases the interest in cryopreservation of these constructs for retrospective investigations, pooling and stock keeping (Malpique et al 2010;Zhang et al 2009).…”