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Mechanical microenvironments and protein expression associated with formation of different skeletal tissues during bone healing.

ABSTRACT: Uncovering the mechanisms of the sensitivity of bone healing to mechanical factors is critical for understanding the basic biology and mechanobiology of the skeleton, as well as for enhancing clinical treatment of bone injuries. This study refined an experimental method of measuring the strain microenvironment at the site of a bone injury during bone healing. This method used a rat model in which a well-controlled bending motion was applied to an osteotomy to induce the formation of pseudarthrosis that is composed of a range of skeletal tissues, including woven bone, cartilage, fibrocartilage, fibrous tissue, and clot tissue. The goal of this study was to identify both the features of the strain microenvironment associated with formation of these different tissues and the expression of proteins frequently implicated in sensing and transducing mechanical cues. By pairing the strain measurements with histological analyses that identified the regions in which each tissue type formed, we found that formation of the different tissue types occurs in distinct strain microenvironments and that the type of tissue formed is correlated most strongly to the local magnitudes of extensional and shear strains. Weaker correlations were found for dilatation. Immunohistochemical analyses of focal adhesion kinase and rho family proteins RhoA and CDC42 revealed differences within the cartilaginous tissues in the calluses from the pseudarthrosis model as compared to fracture calluses undergoing normal endochondral bone repair. These findings suggest the involvement of these proteins in the way by which mechanical stimuli modulate the process of cartilage formation during bone healing.

SUBMITTER: Miller GJ

PROVIDER: S-EPMC5608650 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Mechanical microenvironments and protein expression associated with formation of different skeletal tissues during bone healing.

Miller Gregory J GJ Gerstenfeld Louis C LC Morgan Elise F EF

Biomechanics and modeling in mechanobiology 20150331 6

Uncovering the mechanisms of the sensitivity of bone healing to mechanical factors is critical for understanding the basic biology and mechanobiology of the skeleton, as well as for enhancing clinical treatment of bone injuries. This study refined an experimental method of measuring the strain microenvironment at the site of a bone injury during bone healing. This method used a rat model in which a well-controlled bending motion was applied to an osteotomy to induce the formation of pseudarthros ...[more]

PMID: 25822264

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Project description:Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14 days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (μCT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7 days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis.

Project description:Hedgehog (Hh) signaling is critical in developmental osteogenesis, and recent studies suggest it may also play a role in regulating osteogenic gene expression in the post-natal setting. However, there is a void of studies directly assessing the effect of Hh inhibition on post-natal osteogenesis. This study utilized a cyclic loading-induced ulnar stress fracture model to evaluate the hypothesis that Hh signaling contributes to osteogenesis and angiogenesis during stress fracture healing. Immediately prior to loading, adult rats were given GDC-0449 (Vismodegib - a selective Hh pathway inhibitor; 50mg/kg orally twice daily), or vehicle. Hh signaling was upregulated in response to stress fracture at 3 days (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1 day and 3 days in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1 day, but decreased Shh expression by 37% at 3 days. GDC-0449 decreased woven bone volume (-37%) and mineral density (-17%) at 7 days. Dynamic histomorphometry revealed that the 7 day callus was composed predominantly of woven bone in both groups. The observed reduction in woven bone occurred concomitantly with decreased expression of Alpl and Ibsp, but was not associated with differences in early cellular proliferation (as determined by callus PCNA staining at 3 days), osteoblastic differentiation (Osx expression at 1 day and 3 days), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1 day and 3 days), or bone resorption metrics (callus TRAP staining at 3 days, Rankl and Opg expression at 1 day and 3 days). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3 days, which was associated with increased Hif1α gene expression (+30%). Dynamic histomorphometric analysis demonstrated that GDC-0449 also inhibited lamellar bone formation. Lamellar bone analysis of the loaded limb (directly adjacent to the woven bone callus) showed that GDC-0449 significantly decreased mineral apposition rate (MAR) and bone formation rate (BFR/BS) (-17% and -20%, respectively). Lamellar BFR/BS in the non-loaded ulna was also significantly decreased (-37%), indicating that Hh signaling was required for normal bone modeling. In conclusion, Hh signaling plays an important role in post-natal osteogenesis in the setting of stress fracture healing, mediating its effects directly through regulation of bone formation and angiogenesis.

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Mechanical microenvironments and protein expression associated with formation of different skeletal tissues during bone healing.

Publications

Mechanical microenvironments and protein expression associated with formation of different skeletal tissues during bone healing.

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