Differential growth factor adsorption to calvarial osteoblast-secreted extracellular matrices instructs osteoblastic behavior

Abstract

Craniosynostosis (CS), the premature ossification of cranial sutures, is attributed to increased osteogenic potential of resident osteoblasts, yet the contribution of the surrounding extracellular matrix (ECM) on osteogenic differentiation is unclear. The osteoblast-secreted ECM provides binding sites for cellular adhesion and regulates the transport and signaling of osteoinductive factors secreted by the underlying dura mater. The binding affinity of each osteoinductive factor for the ECM may amplify or mute its relative effect, thus contributing to the rate of suture fusion. The purpose of this paper was to examine the role of ECM composition derived from calvarial osteoblasts on protein binding and its resultant effect on cell phenotype. We hypothesized that potent osteoinductive proteins present during sutural fusion (e.g., bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-β1)) would exhibit distinct differences in binding when exposed to ECMs generated by human calvarial osteoblasts from unaffected control individuals (CI) or CS patients. Decellularized ECMs produced by osteoblasts from CI or CS patients were incubated in the presence of BMP-2 or TGF-β1, and the affinity of each protein was analyzed. The contribution of ECM composition to protein binding was interrogated by enzymatically modulating proteoglycan content within the ECM. BMP-2 had a similar binding affinity for each ECM, while TGF-β1 had a greater affinity for ECMs produced by osteoblasts from CI compared to CS patients. Enzymatic treatment of ECMs reduced protein binding. CS osteoblasts cultured on enzymatically-treated ECMs secreted by osteoblasts from CI patients in the presence of BMP-2 exhibited impaired osteogenic differentiation compared to cells on untreated ECMs. These data demonstrate the importance of protein binding to cell-secreted ECMs and confirm that protein-ECM interactions have an important role in directing osteoblastic differentiation of calvarial osteoblasts.

Figure 1. Overlay images (bright field and fluorescent) of TGF-β1 (0.05 ng/µl) (A, B) and BMP-2 (0.04 ng/µl) (C, D) bound to ECMs produced by CSObs (A, C) and CIObs (B, D).

Images are representative of experiments performed in triplicate and are taken at 100x magnification; scale bars represent 50 µm.

Figure 2. Scatchard plot analysis of TGF-β1 and BMP-2 binding to ECMs secreted by CSObs (A, C) and CIObs (B, D).

Scatchard plot for representative data set in each culture condition is included as inset. Plots are representative of experiments performed in triplicate. K_d represents the dissociation constant.

Figure 3. Alcian Blue stains of GAGs present in ECMs produced by CSObs (A) and CIObs (B) after heparanase (HS), chondroitinase ABC (CABC), both enzymes (BOTH) and without (CONTROL) enzyme treatment.

Images taken at 100X magnification; scale bars represent 50 µm. C: Quantification of Alcian blue stain. *p<0.05 vs. control, #p<0.05 vs. CIObs ECM.

Figure 4. Immunohistochemical staining of molecules contained with ECMs secreted by CSObs (A) and CIObs (B).

From left to right, images for decorin (DCN), biglycan (BGN), collagen type 1 (COLI), syndecan-2 (SYN-2), and fibronectin (FN). Images are taken at 40x magnification; scale bars represent 100 µm.

Figure 5. Quantitative PCR results for genes monitored in CSObs when seeded on enzyme- treated or untreated ECM from CSObs or CIObs or TCP: ALP (A), RUNX2 (B), COL1A (C), DCN (D), BGN (E), and calcium deposition (F).

Values reflect fold change in the target mRNA expression over RPL13. #p<0.05 vs. untreated ECM; *p<0.05 vs. TCP.

Figure 6. Quantitative PCR results for genes monitored in CIObs when seeded on enzyme- treated or untreated ECM from CSObs or CIObs or TCP: ALP (A), RUNX2 (B), COL1A (C), and calcium deposition (D).

Values reflect fold change in the target mRNA expression over RPL13. #p<0.05 vs. untreated ECM; *p<0.05 vs. TCP; $p<0.05 vs. CSObs ECM.

Figure 7. Quantitative PCR results for genes monitored in CSObs seeded on enzyme-treated or untreated ECM from CIObs or TCP control in the presence of TGF-β1 or BMP-2: ALP (A), RUNX2 (B), COL1A (C), and calcium deposition (D).

Values reflect fold change in the target mRNA expression over RPL13. #p<0.05 vs. untreated ECM, *p<0.05 vs. TCP.