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Effects of in vivo mechanical loading on large bone defect regeneration.

ABSTRACT: Fracture healing is highly sensitive to mechanical conditions; however, the effects of mechanical loading on large bone defect regeneration have not been evaluated. In this study, we investigated the effects of functional loading on repair of critically sized segmental bone defects. About 6-mm defects were created in rat femora, and each defect received 5 µg recombinant human bone morphogenetic protein-2 (rhBMP-2), delivered in alginate hydrogel. Limbs were stabilized by either stiff fixation plates for the duration of the study or compliant plates that allowed transfer of compressive ambulatory loads beginning at week 4. Healing was assessed by digital radiography, microcomputed tomography, mechanical testing, histology, and finite element modeling. Loading significantly increased regenerate bone volume and average polar moment of inertia. The response to loading was location-dependent with the polar moment of inertia increased at the proximal end of the defect but not the distal end. As a result, torsional stiffness was 58% higher in the compliant plate group, but failure torque was not altered. In single samples assessed for histology from each group, a qualitatively greater amount of cartilage and a lesser degree of remodeling to lamellar bone occurred in the loaded group compared to the stiff plate group. Finally, principal strain histograms, calculated by FE modeling, revealed that the compliant plate samples had adapted to more efficiently distribute loads in the defects. Together, these data demonstrate that functional transfer of axial loads alters BMP-induced large bone defect repair by increasing the amount and distribution of bone formed within the defect.

SUBMITTER: Boerckel JD

PROVIDER: S-EPMC3307871 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Effects of in vivo mechanical loading on large bone defect regeneration.

Boerckel Joel D JD Kolambkar Yash M YM Stevens Hazel Y HY Lin Angela S P AS Dupont Kenneth M KM Guldberg Robert E RE

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20111214 7

Fracture healing is highly sensitive to mechanical conditions; however, the effects of mechanical loading on large bone defect regeneration have not been evaluated. In this study, we investigated the effects of functional loading on repair of critically sized segmental bone defects. About 6-mm defects were created in rat femora, and each defect received 5 µg recombinant human bone morphogenetic protein-2 (rhBMP-2), delivered in alginate hydrogel. Limbs were stabilized by either stiff fixation pl ...[more]

PMID: 22170172

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Project description:It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 min, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 h between sessions. Ulnae were sampled at 11 time points, from 4 h to 32days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/?-catenin and TGF-? signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading.

Project description:The advent of high-throughput measurements of gene expression and bioinformatics analysis methods offers new ways to study gene expression patterns. The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading, during key periods of the bone formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for three minutes per day, while the left forearm served as a non-loaded, contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct, time-dependent patterns of gene expression were identified over the time course and categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes were then grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1-related genes in the early response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/?-catenin signaling pathway inhibitors in the downregulated gene clusters. Several novel gene groups were identified as well, including chemokine-related genes which were upregulated early but downregulated later in the time course, solute carrier genes which were both up- and downregulated, and muscle-related genes which were primarily downregulated. Time Course with 11 time points, each plus & minus mechanical stimulation with 5 replicates per experimental group (except 12d group which has 4 replicates). Daily mechanical loading was applied to the forearm (24 hours between loading sessions), and ulnae were sampled at indicated time points (4h, 12h, 1d, 2d, 4d, 6d, 8d, 12d, 16d, 24d, or 32d).

Dataset Information

Effects of in vivo mechanical loading on large bone defect regeneration.

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Effects of in vivo mechanical loading on large bone defect regeneration.

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