Matrix Rigidity Induces Osteolytic Gene Expression of Metastatic Breast Cancer Cells

Nearly 70% of breast cancer patients with advanced disease will develop bone metastases. Once established in bone, tumor cells produce factors that cause changes in normal bone remodeling, such as parathyroid hormone-related protein (PTHrP). While enhanced expression of PTHrP is known to stimulate osteoclasts to resorb bone, the environmental factors driving tumor cells to express PTHrP in the early stages of development of metastatic bone disease are unknown. In this study, we have shown that tumor cells known to metastasize to bone respond to 2D substrates with rigidities comparable to that of the bone microenvironment by increasing expression and production of PTHrP. The cellular response is regulated by Rho-dependent actomyosin contractility mediated by TGF-ß signaling. Inhibition of Rho-associated kinase (ROCK) using both pharmacological and genetic approaches decreased PTHrP expression. Furthermore, cells expressing a dominant negative form of the TGF-ß receptor did not respond to substrate rigidity, and inhibition of ROCK decreased PTHrP expression induced by exogenous TGF-ß. These observations suggest a role for the differential rigidity of the mineralized bone microenvironment in early stages of tumor-induced osteolysis, which is especially important in metastatic cancer since many cancers (such as those of the breast and lung) preferentially metastasize to bone

Bone morphogenetic proteins − 7 and − 2 in the treatment of delayed osseous union secondary to bacterial osteitis in a rat model

Background: Bone infections due to trauma and subsequent delayed or impaired fracture healing represent a great challenge in orthopedics and trauma surgery. The prevalence of such bacterial infection-related types of delayed non-union is high in complex fractures, particularly in open fractures with additional extensive soft-tissue damage. The aim of this study was to establish a rat model of delayed osseous union secondary to bacterial osteitis and investigate the impact of rhBMP-7 and rhBMP-2 on fracture healing in the situation of an ongoing infection. Methods: After randomization to four groups 72 Sprague-Dawley rats underwent a transverse fracture of the midshaft tibia stabilized by intramedullary titanium K-wires. Three groups received an intramedullary inoculation with Staphylococcus aureus (103 colony-forming units) before stabilization and the group without bacteria inoculation served as healing control. After 5 weeks, a second surgery was performed with irrigation of the medullary canal and local rhBMP-7 and rhBMP-2 treatment whereas control group and infected control group received sterile saline. After further 5 weeks rats were sacrificed and underwent biomechanical testing to assess the mechanical stability of the fractured bone. Additional micro-CT analysis, histological, and histomorphometric analysis were done to evaluate bone consolidation or delayed union, respectively, and to quantify callus formation and the mineralized area of the callus. Results: Biomechanical testing showed a significantly higher fracture torque in the non-infected control group and the infected rhBMP-7- and rhBMP-2 group compared with the infected control group (p < 0.001). RhBMP-7 and rhBMP-2 groups did not show statistically significant differences (p = 0.57). Histological findings supported improved bone-healing after rhBMP treatment but quantitative micro-CT and histomorphometric results still showed significantly more hypertrophic callus tissue in all three infected groups compared to the non-infected group. Results from a semiquantitative bone-healing-score revealed best bone-healing in the non-infected control group. The expected chronic infection was confirmed in all infected groups. Conclusions: In delayed bone healing secondary to infection rhBMP treatment promotes bone healing with no significant differences in the healing efficacy of rhBMP-2 and rhBMP-7 being noted. Further new therapeutic bone substitutes should be analyzed with the present rat model for delayed osseous union secondary to bacterial osteitis

Designing Bioactive Delivery Systems for Tissue Regeneration

The direct infusion of macromolecules into defect sites generally does not impart adequate physiological responses. Without the protection of delivery systems, inductive molecules may likely redistribute away from their desired locale and are vulnerable to degradation. In order to achieve efficacy, large doses supplied at interval time periods are necessary, often at great expense and ensuing detrimental side effects. The selection of a delivery system plays an important role in the rate of re-growth and functionality of regenerating tissue: not only do the release kinetics of inductive molecules and their consequent bioactivities need to be considered, but also how the delivery system interacts and integrates with its surrounding host environment. In the current review, we describe the means of release of macromolecules from hydrogels, polymeric microspheres, and porous scaffolds along with the selection and utilization of bioactive delivery systems in a variety of tissue-engineering strategies

The effect of polyurethane scaffold surface treatments on the adhesion of chondrocytes subjected to interstitial perfusion culture

The purpose of this study was to measure chondrocytes detachment from cellularized constructs cultured in a perfusion bioreactor, and to evaluate the effect of different scaffold coatings on cell adhesion under a fixed flow rate. The scaffolds were polyurethane foams, treated to promote cell attachment and seeded with human chondrocytes. In a preliminary static culture experiment, the scaffolds were imbibed with fetal bovine serum (FBS) and then cultured for 4 weeks. To quantify cell detachment, the number of detached cells from the scaffold treated with FBS was estimated under different interstitial perfusion flow rates and shear stress levels (0.005 mL/min equivalent to 0.05 mPa, 0.023 mL/min equivalent to 0.23 mPa, and 0.045 mL/min equivalent to 0.45 mPa). Finally, groups of scaffolds differently treated (FBS, plasma plus FBS, plasma plus collagen type I) were cultured under a fixed perfusion rate of 0.009 mL/min, equivalent to a shear stress of 0.09 mPa, and the detached cells were counted. Static cultivation showed that cell proliferation increased with time and matrix biosynthesis decreased after the first week of culture. Perfused culture showed that the number of detached cells increased with the perfusion rate on FBS-treated constructs. The plasma-treated/collagen-coated scaffolds showed the highest resistance to cell detachment. To minimize cell detachment, the perfusion rate must be maintained in the order of 0.02 mL/min, giving a shear stress of 0.2 mPa. Our set-up allowed estimating the resistance to cell detachment under interstitial perfusion in a repeatable manner, to test other scaffold coatings and cell type

Imaging Analysis of the In vivo Bioreactor: A Preliminary Study

The in vivo bioreactor is a hermetically sealed, acellular hydroxyapatite scaffold coated with growth factors that has a pulsating vascular pedicle leash threaded through its center. Tissue-engineered bone is created in weeks while the bioreactor remains embedded under the skin of an animal. The bioreactor also provides a model to study osteogenesis and pathologic scenarios such as tumor progression and metastasis by creating a controlled microenvironment that makes skeletogenesis amenable to genetic and physical manipulation. Animal euthanasia is required to quantitate bioreactor osteogenesis through histomorphometry. Nondestructive measures of new bone growth within the bioreactor are critical to future applications and are the primary questions posed in this study. We compared microcomputed tomography and micro-MRI assessments of bioreactor osteogenesis with conventional histomorphometric measurements in 24 bioreactors and asked if new bone formation could be calculated while the animal was alive. Microcomputed tomography visually, but not numerically, differentiated engineered new bone on its coral scaffold. Dynamic contrast-enhanced micro-MRI demonstrated augmented vascular flow through the bioreactor. Three-dimensional imaging can nondestructively detect tissue-engineered osteogenesis within the implanted bioreactor in vivo, furthering the usefulness of this unique model system