Synthesis and characterization of biomimetic citrate-based biodegradable composites

Abstract

Natural bone apatite crystals, which mediate the development and regulate the load-bearing function of bone, have recently been associated with strongly bound citrate molecules. However, such understanding has not been translated into bone biomaterial design and osteoblast cell culture. In this work, we have developed a new class of biodegradable, mechanically strong, and biocompatible citrate-based polymer blends (CBPBs), which offer enhanced hydroxyapatite binding to produce more biomimetic composites (CBPBHAs) for orthopedic applications. CBPBHAs consist of the newly developed osteoconductive citrate-presenting biodegradable polymers, crosslinked urethane-doped polyester and poly (octanediol citrate), which can be composited with up to 65 wt % hydroxyapatite. CBPBHA networks produced materials with a compressive strength of 116.23 ± 5.37 MPa comparable to human cortical bone (100-230 MPa), and increased C2C12 osterix gene and alkaline phosphatase gene expression in vitro. The promising results above prompted an investigation on the role of citrate supplementation in culture medium for osteoblast culture, which showed that exogenous citrate supplemented into media accelerated the in vitro phenotype progression of MG-63 osteoblasts. After 6 weeks of implantation in a rabbit lateral femoral condyle defect model, CBPBHA composites elicited minimal fibrous tissue encapsulation and were well integrated with the surrounding bone tissues. The development of citrate-presenting CBPBHA biomaterials and preliminary studies revealing the effects of free exogenous citrate on osteoblast culture shows the potential of citrate biomaterials to bridge the gap in orthopedic biomaterial design and osteoblast cell culture in that the role of citrate molecules has previously been overlooked.

Keywords: biodegradable composites; bone tissue engineering; citric acid; osterix.

Figure 1

Material characterizationof CBPBHA composites.(A)DSC thermograms of various CBPBs. (B) In vitro degradation of CBPBHA-90, CBPBHA-100, and CBPBHA-0 composites (PBS; 37 °C) at 1, 2, 4, 8, 12 and 24 weeks of incubation. (C) Compressive modulus of CBPBHA composites. (D) Compressive peak strength of CBPBHA composites. X-axis values represent the total CUPE percentage in CBPBs. All CBPBHA composites were polymerized at 80 °C for 5 days and 120 °C under 2 Pa vacuum for 1 day. Data are expressed as the mean ± S.D. (n = 6) and (* represents significance at p < 0.05).

Figure 2

Representative SEM images of (A) CBPBHA-0, (B) CBPBHA-100, and (C) CBPBHA-90composites mineralized in 4X simulated body fluid (SBF) at 0, 3, and 15 days.(D) EDX analysis of minerals on CBPBHA-90 films incubated in SBF for 15 days. All scale bars are 10 um. The EDX results revealed that the Ca/P ratio of apatite formed on the composites was 1.39 ± 0.25, which is comparable to octacalcium (OCP, Ca/P=1.33) and tricalcium phosphate (TCP, Ca/P=1.5).

Figure 3

In vitro cytocompatibility and osteoblastic differentiation of C2C12s cultured on CBPB-100, CBPBHA-100, and CBPBHA-90 films. (A)Representative SEM images of C2C12s 24h post-seeding.(White arrow indicates the cell monolayer and all scale bars are 100µm).(B)Alkaline phosphatase (ALP) and (C) osterix (Osx) gene expression of C2C12s cultured on CBPB and CBPBHA films.(D) Cumulative citrate release from CBPBand CBPBHA films in PBS at 37 °Cfor 3, 7, and 14 days. Data are expressed as the mean ± S.D. (n = 4).* represents significance at p < 0.05 and # represents p > 0.05.

Figure 4

In vitro MG-63 alkaline phosphatase (ALP) production in citrate-supplemented medium over a 10 day time period(* represents significance at p < 0.05).

Figure 5

Representative 3-D micro-CT and histological images demonstrating the osteointegration and foreign body response, respectively, of CBPBHA-90 and CBPBHA-100 discs implanted into a rabbit lateral femoral condyle after 6 weeks.(A-B)3-D re-constructed micro-CT images, (C-D) H&E stained sections, (E-F) von Kossa stainedsections, and (G-H)toluidine blue stainedsections (white arrow: implants; I: Implant; yellow arrow: mineralization at the interface).

Figure 6

Osteointegration evaluation of CBPBHA-100 and CBPBHA-90 implants. (A) Bone-to-implant contact analysis of CBPBHA-100 and CBPBHA-90 implants at 6 weeks of post-implantation. (B) 2-D micro-CT images of the CBPBHA-90 implants with the surrounding bone of the lateral femoral condyle at 6 weeks of post-implantation (white arrow indicates polymer implant).

Figure 7

Representative images of CD68 and CD163 stained sections demonstrating the foreign body response ofCBPBHA-100 and CBPBHA-90discs implanted into a rabbit lateral femoral condyle defect after 6 weeks. Rat achilles tendon sections were used as a positive control(yellow arrow: brow-stained macrophages).

Scheme 1

Schematic representation of CBPBHA composites.