BMP9 is a potent inducer of chondrogenesis, volumetric expansion and collagen type II accumulation in bovine auricular cartilage chondroprogenitors

Abstract

Reconstruction of the outer ear currently requires harvesting of cartilage from the posterior of the auricle or ribs leading to pain and donor site morbidity. An alternative source for auricular reconstruction is in vitro tissue engineered cartilage using stem/progenitor cells. Several candidate cell-types have been studied with tissue-specific auricular cartilage progenitor cells (AuCPC) of particular interest. Whilst chondrogenic differentiation of competent stem cells using growth factor TGFβ1 produces cartilage this tissue is frequently fibrocartilaginous and lacks the morphological features of hyaline cartilage. Recent work has shown that growth factor BMP9 is a potent chondrogenic and morphogenetic factor for articular cartilage progenitor cells, and we hypothesised that this property extends to cartilage-derived progenitors from other tissues. In this study we show monoclonal populations of AuCPCs from immature and mature bovine cartilage cultured with BMP9 produced cartilage pellets have 3-5-fold greater surface area in sections than those grown with TGFβ1. Increased volumetric growth using BMP9 was due to greater sGAG deposition in immature pellets and significantly greater collagen accumulation in both immature and mature progenitor pellets. Polarised light microscopy and immunohistochemical analyses revealed that the organisation of collagen fibrils within pellets is an important factor in the growth of pellets. Additionally, chondrocytes in BMP9 stimulated cell pellets had larger lacunae and were more evenly dispersed throughout the extracellular matrix. Interestingly, BMP9 tended to normalise the response of immature AuCPC monoclonal cell lines to differentiation cues whereas cells exhibited more variation under TGFβ1. In conclusion, BMP9 appears to be a potent inducer of chondrogenesis and volumetric growth for AuCPCs a property that can be exploited for tissue engineering strategies for reconstructive surgery though with the caveat of negligible elastin production following 21-day treatment with either growth factor.

Fig 1. Brightfield microscopy of toluidine blue and picrosirius red staining of auricular chondroprogenitor cells cultured in high-density pellets for 21 days.

Pellets from three immature and three mature clones (each isolated from different animals) containing 5x10⁵ AuCPCs were cultured in control medium or control medium supplemented with 10ng ml^-1 TGFβ1 or 100ng ml^-1 BMP9 to compare their chondrogenic effect. Metachromatic toluidine blue staining labels the presence of sGAG in cultured cell pellets. Picrosirius red staining shows localisation and organisation of collagen fibrils. Scale bar represents 1000 μm.

Fig 2. Polarised light microscopy of picrosirius red staining of auricular chondroprogenitor cells cultured in high-density pellets for 21 days and native bovine auricular cartilages.

A. Representative images shown from pellets from three immature and three mature clones (each isolated from different animals) containing 5x10⁵ AuCPCs were cultured in control medium or control medium supplemented with 10ng ml^-1 TGFβ1 or 100ng ml^-1 BMP9. Polarised imaging of picrosirius red stained slides showed increased fibril organisation and the formation of concentric rings of fibrils in response to stimulation with TGFβ1, whist BMP9 treatment leads to more anisotropic matrix that is more representative of native tissue. B. Polarised light microscopy of native immature and mature bovine auricular cartilage showing changing collagen fibril organisation and density with age. Scale bars represent 100 μm.

Fig 3. Verhoeff elastic cartilage staining of auricular chondroprogenitor cells cultured in high density pellets for 21 days and native bovine auricular cartilages.

A. Representative images shown for pellets from three immature and three mature clones containing 5x10⁵ AuCPCs cultured in control medium or control medium supplemented with 10ng ml^-1 TGFβ1 or 100ng ml^-1 BMP9. Neither treatment led to the deposition of extracellular elastin fibres, but increased intra/pericellular staining was seen in response to TGFβ1. B. Staining of native bovine auricular tissue shows a decrease in the density and interconnectivity of elastic fibre networks with age. Scale bars represent 100 μm.

Fig 4. Immunohistochemical labelling of aggrecan in extracellular matrix produced by auricular chondroprogenitor cells cultured in high-density pellets for 21 days.

Immunolabelling showed that culture with BMP9 and TGFβ1 and unsupplemented medium demonstrate aggrecan and collagen type II deposition. In contrast, little, if any, collagen type I was found in TGFβ1 stimulated pellets and only deposited in low levels of around the periphery of BMP9 stimulated pellets. The inferior aspect of MP9 treated pellets is shown by an asterisk. Scale bar represents 1000μm.

Fig 5. Quantitative surface area and biochemical analyses of high-density pellets of auricular chondroprogenitors after 21 days in culture.

The surface area of sectioned pellets stained with toluidine blue was quantified using Image J (A; n = 3). Analysis of the sGAG deposited in each of the pellets was performed using the DMMB assay (B; n = 5), the DNA content of each pellet was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (C; n = 5) and the hydroxyproline in each pellet quantified using hydroxyproline assay (E. n = 3–5). Results of sGAG and hydroxyproline are also presented as normalised to the DNA content of each pellet (D. and F.). Samples in which no hydroxyproline was detected are labelled ‘n.d.’. Significance was taken as p<0.05, ‘a’ denotes a significant difference to ‘Immature control, ‘b’ to ‘Mature control’, ‘c’ to ‘Immature TGFβ1’, ‘d’ to Mature TGFβ1’, ‘e’ to Immature BMP9’ and ‘f’ to ‘Mature BMP9’.

Fig 6. Quantitative gene expression of auricular chondroprogenitor cells cultured in highdensity pellets for 21 days.

Pellets from one immature (n = 4) and one mature clone (n = 4) containing 5x10⁵ AuCPCs were cultured in control medium or control medium supplemented with 10ng ml^-1 TGFβ1 or 100ng ml^-1 BMP9. Cells from each donor were also collected at the point of seeding as a ‘Day 0’ time point (n = 3). Gene expression of A. ACAN, B. COL1A1, C. COL2A1, D. ELN and E. SOX9 were determined using reverse transcription quantitative real-time PCR. Significance was taken as p<0.05, ‘a’ denotes a significant difference to ‘Immature Day 0’, ‘b’ to ‘Mature Day 0’, ‘c’ to ‘Immature Control, ‘d’ to ‘Mature Control’, ‘e’ to ‘Immature TGFβ1’, ‘f’ to Mature TGFβ1’, ‘g’ to Immature BMP9’ and ‘h’ to ‘Mature BMP9’.