Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

Abstract

Cell therapies for articular cartilage defects rely on expanded chondrocytes. Mesenchymal stem cells (MSC) represent an alternative cell source should their hypertrophic differentiation pathway be prevented. Possible cellular instruction between human articular chondrocytes (HAC) and human bone marrow MSC was investigated in micromass pellets. HAC and MSC were mixed in different percentages or incubated individually in pellets for 3 or 6 weeks with and without TGF-β1 and dexamethasone (±T±D) as chondrogenic factors. Collagen II, collagen X and S100 protein expression were assessed using immunohistochemistry. Proteoglycan synthesis was evaluated applying the Bern score and quantified using dimethylmethylene blue dye binding assay. Alkaline phosphatase activity (ALP) was detected on cryosections and soluble ALP measured in pellet supernatants. HAC alone generated hyaline-like discs, while MSC formed spheroid pellets in ±T±D. Co-cultured pellets changed from disc to spheroid shape with decreasing number of HAC, and displayed random cell distribution. In -T-D, HAC expressed S100, produced GAG and collagen II, and formed lacunae, while MSC did not produce any cartilagespecific proteins. Based on GAG, collagen type II and S100 expression chondrogenic differentiation occurred in -T-D MSC co-cultures. However, quantitative experimental GAG and DNA values did not differ from predicted values, suggesting only HAC contribution to GAG production. MSC produced cartilage-specific matrix only in +T+D but underwent hypertrophy in all pellet cultures. In summary, influence of HAC on MSC was restricted to early signs of neochondrogenesis. However, MSC did not contribute to the proteoglycan deposition, and HAC could not prevent hypertrophy of MSC induced by chondrogenic stimuli.

Osteogenic Differentiation of Mesenchymal Stem Cells by Mimicking the Cellular Niche of the Endochondral Template

“…However, unlike previous studies, there was also an increase in late osteogenic markers (calcium) compared with the control group (osteogenic). Importantly, and in contrast to previous studies, 37,[40][41][42][43][44][46][47][48][49]69 this increase in ALP and calcium content was achieved in a 3D scaffoldless construct, without the use of any osteogenic supplements. When compared with the calcium content results, 49 the increase in calcium content in the coculture group with added MSCs/ HUVECs, but no osteogenic supplements, was significantly higher than in the same group cultured in osteogenic supplements as reported by Freeman et al 49 There was significantly higher calcium content in the group cultured with osteogenic supplements than in the group without Quantitative analysis of cross-sectional area of the rudimentary vessels present in the aggregates.…”

“…Moreover, similar to previous studies, 37,[40][41][42][43][44][46][47][48][49]69 the coculture group with both HUVECs and MSCs added had significantly higher ALP production than the control groups (osteogenic and noncoculture group) after 3 weeks of coculture. However, unlike previous studies, there was also an increase in late osteogenic markers (calcium) compared with the control group (osteogenic).…”

“…[42][43][44][45] The osteogenic potential of MSC/chondrocyte or osteoblast/ chondrocyte cocultures has been variable and inconclusive in both two-dimensional (2D) and 3D cultures. [46][47][48] One study investigated the effect of coculture of hMSCs and chondrocytes, without the use of osteogenic supplements, and found that there was no ALP production/ expression in 3D aggregate culture. 47 However, direct 2D coculture of rat osteoblasts and bovine chondrocytes reported a significant increase in ALP production over a period of 6 weeks and there was significantly higher ALP activity in the coculture group than the osteoblast group alone.…”

“…[46][47][48] One study investigated the effect of coculture of hMSCs and chondrocytes, without the use of osteogenic supplements, and found that there was no ALP production/ expression in 3D aggregate culture. 47 However, direct 2D coculture of rat osteoblasts and bovine chondrocytes reported a significant increase in ALP production over a period of 6 weeks and there was significantly higher ALP activity in the coculture group than the osteoblast group alone. 48 Coculture of MSCs and endothelial cells through transwell inserts induced MSCs to undergo both osteogenesis and chondrogenesis, through the endothelin-1 phosphatidylinositol 3-kinase/AKT (AKT) signaling pathway.…”

Les cellules souches en ingénierie des tissus ostéoarticulaires et vasculaires

“…La différenciation chondrogé-nique des CSM, comme dans l'ossification endochondrale, ne s'arrête pas au stade du chondrocyte mature mais se poursuit vers l'hypertrophie du chondrocyte, la minéralisation de la MEC et la formation d'os [10]. Afin de prévenir le risque de formation d'un tissu minéralisé notamment in vivo , il semble cependant possible de contrôler ce processus en cocultivant les CSM avec des chondrocytes articulaires [11]. Par ailleurs, l'hypoxie, outre son action stimulatrice sur la différenciation chondro génique précoce des CSM, pourrait également être utilisée pour prévenir l'apparition de marqueurs du stade terminal de différenciation chondrogénique [12].…”