Maureen E. Lynch

Mesenchymal stem cells and insulin‐like growth factor‐I gene‐enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin‐like growth factor‐I (IGF‐I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF‐I gene‐enhanced MSCs (AdIGF‐MSCs) on tendon healing in vivo. Collagenase‐induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 × 106 MSCs or 10 × 106 AdIGF‐MSCs. Control limbs were injected with 1 mL of phosphate‐buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF‐I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase‐3 (MMP‐3), matrix metalloproteinase‐13 (MMP‐13), and aggrecanase‐1 (ADAMTS‐4) were similar in MSC and control tendons. Both MSC and AdIGF‐MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF‐MSCs for the treatment of tendinitis.

Engineered culture models for studies of tumor-microenvironment interactions.

Heterogeneous microenvironmental conditions play critical roles in cancer pathogenesis and therapy resistance and arise from changes in tissue dimensionality, cell-extracellular matrix (ECM) interactions, soluble factor signaling, oxygen as well as metabolic gradients, and exogeneous biomechanical cues. Traditional cell culture approaches are restricted in their ability to mimic this complexity with physiological relevance, offering only partial explanation as to why novel therapeutic compounds are frequently efficacious in vitro but disappoint in preclinical and clinical studies. In an effort to overcome these limitations, physical sciences-based strategies have been employed to model specific aspects of the cancer microenvironment. Although these strategies offer promise to reveal the contributions of microenvironmental parameters on tumor initiation, progression, and therapy resistance, they, too, frequently suffer from limitations. This review highlights physicochemical and biological key features of the tumor microenvironment, critically discusses advantages and limitations of current engineering strategies, and provides a perspective on future opportunities for engineered tumor models.

Tibial compression is anabolic in the adult mouse skeleton despite reduced responsiveness with aging

The ability of the skeleton to adapt to mechanical stimuli diminishes with age in diaphyseal cortical bone, making bone formation difficult for adults. However, the effect of aging on adaptation in cancellous bone, tissue which is preferentially lost with age, is not well characterized. To develop a model for early post-menopausal women and determine the effect of aging on cancellous bone adaptation in the adult mouse skeleton, in vivo tibial compression was applied to adult (26 week old) osteopenic female mice using loading parameters, peak applied load and peak diaphyseal strain magnitude, that were previously found to be osteogenic in young, growing (10 week old) mice. A Load-Matched group received the same peak applied loads (corresponding to +2100 με at the medial diaphysis of the tibia) and a Strain-Matched group received the same peak diaphyseal strains (+1200 με, requiring half the load) as the young mice. The effects of mechanical loading on bone mass and architecture in adult mice were assessed using micro-computed tomography and in vivo structural stiffness measures. Adaptation occurred only in the Load-Matched group in both the metaphyseal and diaphyseal compartments. Cancellous bone mass increased 54% through trabecular thickening, and cortical area increased 41% through medullary contraction and periosteal expansion. Adult mice were able to respond to an anabolic stimulus and recover bone mass to levels seen in growing mice; however, the adaptive response was reduced relative to that in 10 week old female mice for the same applied load. Using this osteogenic loading protocol, other factors affecting pathological bone loss can be addressed using an adult osteopenic mouse model.

Cancellous bone adaptation to tibial compression is not sex dependent in growing mice

Mechanical loading can be used to increase bone mass and thus attenuate pathological bone loss. Because the skeletons adaptive response to loading is most robust before adulthood, elucidating sex-specific responses during growth may help maximize peak bone mass. This study investigated the effect of sex on the response to controlled, in vivo mechanical loading in growing mice. Ten-week-old male and female C57Bl/6 mice underwent noninvasive compression of the left tibia. Peak loads of -11.5 N were applied, corresponding to +1,200 microepsilon at the tibial midshaft in both sexes. Cancellous bone mass, architecture, and dynamic formation in the proximal metaphysis were compared between loaded and control limbs via micro-computed tomography and histomorphometry. The strain environment of the proximal metaphysis during loading was characterized using finite element analysis. Both sexes responded to tibial compression through increased bone mass and altered architecture. Cancellous bone mass and tissue density were enhanced in loaded limbs relative to control limbs in both sexes through trabecular thickening and reduced separation. Changes in mass were due to increased cellular activity in loaded limbs compared with control limbs. Adaptation to loading increased the proportion of load transferred by the cancellous bone in the proximal metaphysis. For all cancellous measures, the response to tibial compression did not differ between male and female mice. When similar strains are engendered in males and females, the adaptive response in cancellous bone to mechanical loading does not depend on sex.

In Vivo Tibial Compression Decreases Osteolysis and Tumor Formation in a Human Metastatic Breast Cancer Model

Bone metastasis, the leading cause of breast cancer‐related deaths, is characterized by bone degradation due to increased osteoclastic activity. In contrast, mechanical stimulation in healthy individuals upregulates osteoblastic activity, leading to new bone formation. However, the effect of mechanical loading on the development and progression of metastatic breast cancer in bone remains unclear. Here, we developed a new in vivo model to investigate the role of skeletal mechanical stimuli on the development and osteolytic capability of secondary breast tumors. Specifically, we applied compressive loading to the tibia following intratibial injection of metastatic breast cancer cells (MDA‐MB231) into the proximal compartment of female immunocompromised (SCID) mice. In the absence of loading, tibiae developed histologically‐detectable tumors with associated osteolysis and excessive degradation of the proximal bone tissue. In contrast, mechanical loading dramatically reduced osteolysis and tumor formation and increased tibial cancellous mass due to trabecular thickening. These loading effects were similar to the baseline response we observed in non‐injected SCID mice. In vitro mechanical loading of MDA‐MB231 in a pathologically relevant 3D culture model suggested that the observed effects were not due to loading‐induced tumor cell death, but rather mediated via decreased expression of genes interfering with bone homeostasis. Collectively, our results suggest that mechanical loading inhibits the growth and osteolytic capability of secondary breast tumors after their homing to the bone, which may inform future treatment of breast cancer patients with advanced disease.

In vivo tibial stiffness is maintained by whole bone morphology and cross-sectional geometry in growing female mice

Whole bone morphology, cortical geometry, and tissue material properties modulate skeletal stresses and strains that in turn influence skeletal physiology and remodeling. Understanding how bone stiffness, the relationship between applied load and tissue strain, is regulated by developmental changes in bone structure and tissue material properties is important in implementing biophysical strategies for promoting healthy bone growth and preventing bone loss. The goal of this study was to relate developmental patterns of in vivo whole bone stiffness to whole bone morphology, cross-sectional geometry, and tissue properties using a mouse axial loading model. We measured in vivo tibial stiffness in three age groups (6, 10, 16 wk old) of female C57Bl/6 mice during cyclic tibial compression. Tibial stiffness was then related to cortical geometry, longitudinal bone curvature, and tissue mineral density using microcomputed tomography (microCT). Tibial stiffness and the stresses induced by axial compression were generally maintained from 6 to 16 wks of age. Growth-related increases in cortical cross-sectional geometry and longitudinal bone curvature had counteracting effects on induced bone stresses and, therefore, maintained tibial stiffness similarly with growth. Tissue mineral density increased slightly from 6 to 16 wks of age, and although the effects of this increase on tibial stiffness were not directly measured, its role in the modulation of whole bone stiffness was likely minor over the age range examined. Thus, whole bone morphology, as characterized by longitudinal curvature, along with cortical geometry, plays an important role in modulating bone stiffness during development and should be considered when evaluating and designing in vivo loading studies and biophysical skeletal therapies.

Biomechanical forces in the skeleton and their relevance to bone metastasis: biology and engineering considerations

Bone metastasis represents the leading cause of breast cancer related-deaths. However, the effect of skeleton-associated biomechanical signals on the initiation, progression, and therapy response of breast cancer bone metastasis is largely unknown. This review seeks to highlight possible functional connections between skeletal mechanical signals and breast cancer bone metastasis and their contribution to clinical outcome. It provides an introduction to the physical and biological signals underlying bone functional adaptation and discusses the modulatory roles of mechanical loading and breast cancer metastasis in this process. Following a definition of biophysical design criteria, in vitro and in vivo approaches from the fields of bone biomechanics and tissue engineering that may be suitable to investigate breast cancer bone metastasis as a function of varied mechano-signaling will be reviewed. Finally, an outlook of future opportunities and challenges associated with this newly emerging field will be provided.

Load-induced changes in bone stiffness and cancellous and cortical bone mass following tibial compression diminish with age in female mice

The vertebrate skeleton is an adaptive structure that responds to mechanical stimuli by increasing bone mass under increased mechanical loads. Although experimental animal models have shown the anabolic cortical bone response to applied load decreases with age, no consensus exists regarding whether this adaptive mechanism is affected by age in cancellous bone, the tissue most impacted by age-related bone loss. We used an established murine in vivo tibial loading model to characterize the load-induced cancellous, cortical and whole-bone responses to mechanical stimuli in growing and mature female mice at 6, 10 and 16 weeks of age. The effects of applied load on tibial morphology and stiffness were determined using microcomputed tomography and in vivo bone strains measured at the medial tibial midshaft during applied loading. At all ages, 2 weeks of applied load produced larger midshaft cortical cross-sectional properties (+13–72%) and greater cancellous bone volume (+21–107%) and thicker trabeculae (+31–68%) in the proximal metaphyses of the loaded tibiae. The relative anabolic response decreased from 6 to 16 weeks of age in both the cancellous and cortical envelopes. Load-induced tibial stresses decreased more in 6-week-old mice following loading, which corresponded to increased in vivo tibial stiffness. Stiffness in the loaded tibiae of 16-week-old mice decreased despite moderately increased cortical cross-sectional geometry, suggesting load-induced changes in bone material properties. This study shows that the cancellous and cortical anabolic responses to mechanical stimuli decline with age into adulthood and that cortical cross-sectional geometry alone does not necessarily predict whole-bone functional stiffness.

Three-Dimensional Mechanical Loading Modulates the Osteogenic Response of Mesenchymal Stem Cells to Tumor-Derived Soluble Signals.

Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis

Significance Hydroxyapatite (HA) nanocrystals are key constituents of the bone extracellular matrix and thus likely to influence the pathogenesis of breast cancer skeletal metastasis. However, there is currently an insufficient understanding of HA nanocrystal properties at sites prone to bone metastasis formation. Here we report a novel application of X-ray scattering and Raman imaging to characterize HA nanostructure in mouse models of breast cancer. Our results suggest that bone regions linked with the initiation of metastasis contain less-mature HA nanocrystals and that mammary tumors enhance HA nanocrystal immaturity in these regions even prior to secondary tumor formation. Insights from this work will significantly advance the development of mineralized culture models to investigate how the bone microenvironment regulates breast cancer metastasis. Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.