High-Throughput Screening of Thiol-ene Click Chemistries for Bone Adhesive Polymers

Abstract

Metal surgical pins and screws are employed in millions of orthopedic surgical procedures every year worldwide, but their usability is limited in the case of complex, comminuted fractures or in surgeries on smaller bones. Therefore, replacing such implants with a bone adhesive material has long been considered an attractive option. However, synthesizing a biocompatible bone adhesive with a high bond strength that is simple to apply presents many challenges. To rapidly identify candidate polymers for a biocompatible bone adhesive, we employed a high-throughput screening strategy to assess human mesenchymal stromal cell (hMSC) adhesion toward a library of polymers synthesized via thiol-ene click chemistry. We chose thiol-ene click chemistry because multifunctional monomers can be rapidly cured via ultraviolet (UV) light while minimizing residual monomer, and it provides a scalable manufacturing process for candidate polymers identified from a high-throughput screen. This screening methodology identified a copolymer (1-S2-FT01) composed of the monomers 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) and pentaerythritol tetrakis (3-mercaptopropionate) (PETMP), which supported highest hMSC adhesion across a library of 90 polymers. The identified copolymer (1-S2-FT01) exhibited favorable compressive and tensile properties compared to existing commercial bone adhesives and adhered to bone with adhesion strengths similar to commercially available bone glues such as Histoacryl. Furthermore, this cytocompatible polymer supported osteogenic differentiation of hMSCs and could adhere 3D porous polymer scaffolds to the bone tissue, making this polymer an ideal candidate as an alternative bone adhesive with broad utility in orthopedic surgery.

Keywords: bone adhesive; click chemistry; cytocompatibility; high-throughput screening; orthopedics.

Figure 1

High-throughput screening of thiol–ene polymers via microarrays. (A) Quantification of the number of DAPI-stained hMSC nuclei on each copolymer. The grand mean was computed, and those copolymers whose counts were greater than 1.5 standard deviations above the grand mean are shown on the left of the orange line. The histogram shows the mean for each polymer chemistry ± standard deviation (n = 6 per chemistry). (B) Fluorescent imaging of lead copolymers on a microarray shows F-actin (green) and nuclei (blue). (C) Chemical structures of pentaerythritol allyl ether (PEAE (ET01)), 2,4,6-triallyloxy-1,3,5-triazine (TATA (ET02)), TATATO (FT01), trimethylolpropane ethoxylate triacrylate (TMPETA (AT07-L)), trimethylolpropane tris(3-mercaptopropionate) (TMPTMP (S1)), and PETMP (S2) monomers used for synthesis of the top 5-ranked hMSC adhesive copolymers. (D) hMSC count and viability on candidate copolymers vs. TCP controls (n = 8; ****p < 0.0001). (E) Phase microscopy images of hMSCs seeded on 1-S2-FT01 (TATATO/PETMP) and the TCP control for 24 and 48 h.

Figure 2

1-S2-FT01 resin mechanical characterization and bone adhesion tests. (A) Rheological characterization of 1-S2-FT01 curing when exposed to UV light (3 min UV light, 20 mW cm^–2). (B) Compressive and (C) tensile properties of dry and SBF-soaked 1-S2-FT01 samples. (n = 5; **p < 0.01). (D) 1-S2-FT01 adhesion to bovine tibia segments. The representative image of the polymer adhered to the bone (left) and lap-shear tests of dry and SBF-soaked 1-S2-FT01 to the bone (right) (n = 6; **p < 0.01).

Figure 3

In vitro cytocompatibility and osteogenic differentiation analyses. (A) Representative confocal microscopy images of hMSCs cultured on 1-S2-FT01 and TCP in growth (left) and differentiation (right) media (Hoechst and ActinRed staining). (B) Relative metabolic activity of hMSCs cultured in proliferation (growth) and osteogenic (osteo) media, seeded directly on 1-S2-FT01 and TCP, as the control (n = 6). (C) Representative confocal microscopy images of hMSCs cultured on 1-S2-FT01 and TCP in growth (left) and differentiation (right) media (calcein and CellTracker deep red staining). (D) ALP expression normalized on DNA content (n = 6; **p < 0.01).

Figure 4

1-S2-FT01 adhesion to star- and linear-PCL scaffolds. (A) Representative image of lap-shear adhesion testing of 1-S2-FT01 to a linear-PCL scaffold (dry) (left). For dry and SBF-soaked 1-S2-FT01 to PCL scaffold constructs, stress versus displacement curves of lap-shear adhesion tests (top right) and maximum strength achieved in lap-shear tests (bottom right, n = 6; **p < 0.01). (B) Representative scanning electron microscopy (SEM) images of 1-S2-FT01/linear-PCL scaffold adhesion showing the construct’s surface (top row) and the cross-section (bottom row).