Date Published: July 15, 2017
Author(s): Jingwei Zhang, Melis T. Dalbay, Xiaoman Luo, Erik Vrij, Davide Barbieri, Lorenzo Moroni, Joost D. de Bruijn, Clemens A. van Blitterswijk, J. Paul Chapple, Martin M. Knight, Huipin Yuan.
The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications.
The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation.
Calcium phosphate (CaP) ceramics are widely used in orthopedic, dental and maxillofacial surgery as bone substitutes because of their chemical homology to native bone mineral, excellent biocompatibility and the ability to support osteogenesis on their surface (i.e. osteoconductivity) , , . However, osteoinductivity of bone graft substitutes, i.e. the ability to positively induce osteogenic differentiation of stem cells to form bone, is required for bone regeneration in critical-sized bone defects , . The most common approach to make CaP ceramics osteoinductive is to combine them with growth factors (e.g. bone morphogenetic proteins, BMPs) . However, the cost and safety of such approaches pose major concerns .
Chemical design of materials and application of biological molecules are often used to achieve specific biological responses in tissue regeneration. However there is an increasing amount of evidence suggesting that physical, mechanical or topographical properties of biomaterials also play a pivotal role in controlling biological functions . In particular, it has been reported that the micro- and nano-structured surfaces of biomaterials can mediate cellular behavior including adhesion, morphology, proliferation and differentiation in vitro, . Here, we reported that surface structure or topography of TCP ceramics affect hBMSC morphology, primary cilia expression and ciliary recruitment of p-TGFβR II in vitro. Furthermore, these differences were associated with regulation of osteogenesis. Thus submicron scaled surface features (TCP-S) induced greater cell spreading (Fig. 2), increased primary cilia expression, cilia elongation and recruitment of p-TGFβR II into the cilium (Fig. 3, Fig. 4) associated with increased osteogenic differentiation at both protein (Fig. 5) and gene level (Fig. 6). Furthermore, following an ectopic implantation, this material (TCP-S) also gave rise to heterotopic bone formation in muscle while TCP ceramic implants with micron scaled surface structure (TCP-B) did not (Fig. 7). These findings suggest for the first time, that the topographical cues may drive osteogenic differentiation by modulating primary cilia structure and ciliary recruitment of p-TGFβR II, which is required to activate TGFβ signalling.
In this study we compared TCP ceramics with micron or sub-micron scaled surface structure, termed TCP-B and TCP-S respectively. TCP ceramic with a submicron scale surface induced a more spread stem cell morphology, increased expression and length of primary cilia, recruitment of p-TGFβ RII to the ciliary axoneme and osteogenic differentiation at a cellular and molecular level without any additional osteogenic factors in vitro. Furthermore, this osteogenic response was associated with increased inductive bone formation in vivo. These data not only highlight the importance of topography in regulating osteogenesis but also imply a novel mechanism involving primary cilia elongation and recruitment of p-TGFβ RII to the ciliary axoneme. This may therefore represent a new biomaterial based ‘ciliotherapy’ for use in orthopedic, dental and maxillofacial surgery applications.
All primary cilia and TGFβ experimental work was conducted by MD with support from PC and MK at Queen Mary University of London. The preparation and characterization of TCP, the in vitro analysis of ALP and osteogenic gene expression was conducted by JZ with support from XL, EV, DB, LM and HY at Maastricht University. In vivo analysis was conducted by HY at Sichuan University. JB, CB provided ideas. JZ, MD, MK, and HY conceived the study and wrote the paper. All authors were involved in analysis of different aspects of the results.