PLLA-Nanodiamond Composites and Their Application In Bone Tissue Engineering

摘要

Nanodiamond (ND) is an attractive nanomaterial for reinforcement of polymers [1] due to the ND's superior mechanical and chemical properties, and low biotoxicity. A novel composite material has been produced for bone scaffolds utilizing the biodegradable polymer, poly(L-lactic acid) (PLLA), and octadecylamine-functionalized nanodiamond (ND-ODA) [2]. Composites were prepared by admixing to a PLLA/chloroform solution chloroform suspensions of ND-ODA at concentrations of 0, 1, 3, 5, 7, and 10 (w/w).rnDispersion of ND-ODA in the composites was studied by transmission electron microscopy (TEM). The lower-resolution TEM micrograph of 1% wt ND-ODA/PLLA film (Fig. la) shows nanodiamond particles dispersed in PLLA film on amorphous carbon support. Due to long hydrocarbon chains of ODA the ND-ODA particles have good wettability with the PLLA so there is no segregation of ND-ODA and PLLA, and the polymer completely surrounds the particles. The high-resolution TEM image (Fig. lb) shows ND crystals with attached organic material that can be ODA or PLLA.rnNanoindentation tests show that the mechanical strength of ND-ODA/PLLA composites improves upon addition of ND (Table 1). Even at low concentrations (1% wt) the ND-ODA increased the hardness of the composite by 60% and Young's modulus by 20% over neat PLLA. Based on our preliminary observations, we conclude that further additions of ND-ODA resulted in smaller changes with subsequent saturation in the mechanical properties at ~7% wt (see Table 1).rnFig.l. TEM images of 1 % wt ND-ODA/PLLA: (a) shows ND-ODA with PLLA where the PLLA forms an amorphous film; high-resolution TEM (b) shows thin amorphous layer around the ND particles.rnND is relatively novel nanomaterial. Establishing its biocompatibility requires further studies, especially for modified ND. We studied the biocompatibility of 5-10nm ND and ND-ODA in experiments with a murine osteoblast cell line (7F2, from ATCC). Incubation of a cultured osteoblasts with l-100ug/ml of ND or ND-ODA particles for 4 hours did not show much influence on the cell viability (Fig. 2), as inferred from an alamarBlue™ assay.rnTable 1. Mechanical properties of ND-ODA/PLLA samples determined by nanoindentation.rnFig.2. Cell viability assay (alamarBlueTM based) of 7F2 mouse osteoblasts grown on tissue culture plastic in the absence (TCP), or presence of respectively, 1-100 fig/ml of ND-ODA, and 1-100 Hg/ml ofND. Y-error bars represent the standard deviation for each sample (n=6). *=Statistically significant differences (p<0.05) compared to each two samples by one-way ANOVA with Tukey-Cramer post-tests for multiple comparisons.rnTo test the feasibility of ND-ODA/PLLA as a matrix material supporting cell growth, osteoblasts were cultured on the composites for 6 days. The attachment and proliferation of 7F2 cells on the scaffolds were assessed, respectively, by fluorescent nuclear staining with Hoechst 33258 and thernalamarBlueTM assay. Our results showed that the addition of ND-ODA had only a negligibly small effect on cell proliferation, which is indicative of good biocompatibility of the composites (Fig. 3). The morphology of 7F2 cells growing on all ND-ODA/PLLA composite scaffolds was assessed by SEM. The data (not shown) confirm that the osteoblasts spread on the scaffolds similar to their spreading on TCP (tissue culture plastic).rnFig.3. Normalized increase in alamarBlue™ readings over the 6 days in vitro 7F2 cell culture on pure PLLA, ND-ODA/PLLA (1-10% wt of ND-ODA), and control glass and TCP substrates. Metabolic activities as measured by alamarBlue™ assay every three davs following the 24h seeding period. The data were normalized to the alamarBlue™ fluorescence reading at day 0. Data are plotted as means, Y-error bars represent the standard deviation for each sample (n=3). *= P<0.05; **= P<0.01, values are significantly different from those on glass and TCP substrates at day 6.rnTo summarize, the improved mechanical properties of the PLLA/ND-ODA composites and their good biocompatibility suggest that these materials may be suitable for applications in musculoskeletal tissue engineering.