Temporal metabolic profiling of bone healing in a caprine tibia segmental defect model

Abstract

Bone tissue engineering is an emerging field of regenerative medicine, with a wide array of biomaterial technologies and therapeutics employed. However, it is difficult to objectively compare these various treatments during various stages of tissue response. Metabolomics is rapidly emerging as a powerful analytical tool to establish broad-spectrum metabolic signatures for a target biological system. Developing an effective biomarker panel for bone repair from small molecule data would provide an objective metric to readily assess the efficacy of novel therapeutics in relation to natural healing mechanisms. In this study we utilized a large segmental bone defect in goats to reflect trauma resulting in substantial volumetric bone loss. Characterization of the native repair capacity was then conducted over a period of 12 months through the combination of standard (radiography, computed tomography, histology, biomechanics) data and ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) metabolic profiling. Standard metrics demonstrated that samples formed soft callus structures that later mineralized. Small molecule profiles showed distinct temporal patterns associated with the bone tissue repair process. Specifically, increased lactate and amino acid levels at early time points indicated an environment conducive to osteoblast differentiation and extracellular matrix formation. Citrate and pyruvate abundances increased at later time points indicating increasing mineral content within the defect region. Taurine, shikimate, and pantothenate distribution profiles appeared to represent a shift toward a more homeostatic remodeling environment with the differentiation and activity of osteoclasts offsetting the earlier deposition phases of bone repair. The generation of a comprehensive metabolic reference portfolio offers a potent mechanism for examining novel biomaterials and can serve as guide for the development of new targeted therapeutics to improve the rate, magnitude, and quality of bone regeneration.

Keywords: biomarkers; bone remodeling; bone tissue engineering; caprine; large animal model; metabolomics; regenerative medicine; veterinary research.

Figure 1

Representative radiographic imaging of defects over the study period demonstrating an increase in mineralization and overall remodeling as a factor of time.

Figure 2

Bone mineral density data as a factor of time generated from DEXA analysis.

Figure 3

Representative CT 3D renderings for samples at 3-, 6-, 9-, and 12-month time points. CT renderings are displayed in medial facing (A, C, E, G) and posterior facing (B, D, F, H) angles to highlight mineral content at the site.

Figure 4

Plot of load-to-failure forces for samples at 3-, 6-, 9-, and 12-month time points. Significant differences observed between 3-month and both 9-month and 12-month samples are denoted by * and ** respectively.

Figure 5

Representative Von Kossa histological staining and plotted mineralization data.

Figure 6

Representative Goldner's Trichrome histological staining and plotted osteoid content data.

Figure 7

2D (A) and 3D PLS-DA (B) plots showing separation of small molecule profiles for samples with groups color coded to designate 3, 6, 9, and 12-month groups. VIP score chart (C) illustrating metabolites driving group separation across study period.

Figure 8

Heatmap illustrating abundance profile fluctuations in detected metabolites over study time points. From left to right, columns denote 3, 6, 9, and 12-month time points, with each row representing a detected metabolite.

Figure 9

Histogram depicting normalized metabolite abundances for essential, non-essential, and conditionally essential amino acids detected in all samples.

Figure 10

Diagram depicting time-dependent small molecule normalized abundances as related to glycolysis. (1) Early enhanced lactate levels are indicative of conditions conducive to osteoblastic differentiation and activity. (2) Early enhanced levels of arginine may be indicative of angiogenic activity through the production of nitric oxide. (3) Early enhanced proline levels are indicative of collagen deposition to form soft callus tissue. (4) Increasing citrate/isocitrate and pyruvate levels are indicative of a switch to the TCA cycle for energy production and subsequent mineralization of tissue. (5) Decreasing taurine levels are indicative of more conducive environment for osteoclastic differentiation and may represent a move toward more homeostatic conditions.