Laura R. McCabe - US grants

0074439
Crimp

The objective of this research project is to create a novel, synthetic, biocompatible osteogenic bone substitute having mechanical properties similar to cortical bone. Every year in the U.S. alone, approximately 100,000 bone grafts are performed. Applications for bone substitutes include massive bone loss from neoplasia, comminuted fracture repair, spinal surgery, and fracture treatment in the elderly. The model bone substitute is 1) osteogenic (direct bone formation via transplanted live bone cells called osteoblasts), 2) osteoinductive (the ability to recruit bone forming cells), 3) osteoconductive (the scaffold supports bone in-growth), 4) mechanically stable (able to carry the load of the natural bone) and 5) readily available. Both the limited supply and diminished properties of autografts and allografts have led researchers on a quest for an optimum synthetic bone substitute. Because the mineral content of bone has a composition and crystal structure closely matching hydroxyapatite (HA), it is natural to first turn to bioceramics such as HA-based materials for bone implants. While dense bioceramics do not exhibit osteogenic or osteoinductive properties, HA is osteoconductive. When using HA, the surgeon is faced with a trade-off between the mechanical strength provided by dense ceramics, and the biological incorporation (bone in-growth) provided by porous ceramics. The lack of mechanical strength in combination with the ability to support bone in-growth has prevented HA, or any other bioceramic, from receiving wide spread acceptance as a cancellous or a cortical bone substitute. The research project uses Professor Crimp's background in ceramic processing to create a strong, yet porous HA-based bone substitute where osteogenic and osteoinductive properties are added by in vitro culture of these synthetic bone scaffolds with osteoblasts following protocols developed by the co-PI, Professor McCabe. HA whisker reinforced porous HA and biphasic calcium phosphate (BCP) ceramics will be fabricated using a modified foaming technique. BCP is a mixture of HA and beta-tri-calcium phosphate and is currently receiving attention as a potential bone substitute material. Of fundamental significance is the understanding of the linkages between bioceramic processing and the in vivo behavior by looking at osteoblast adhesion in vitro.

This research project will concentrate on tailoring the processing protocol for whisker reinforced ceramic materials for use as bone replacements to yield improvements in strength, at a fixed porosity level matching that of human cortical bone, while maintaining the inherent biocompatibility of the implant materials. These implants will be the only viable synthetic alternative (because of improved mechanical and osteogenic properties) to currently available graft materials for repair of damaged cortical bone tissue.

[unreadable] DESCRIPTION (provided by applicant): Type I diabetics are at risk for bone loss and, with an increasing lifespan, are susceptible to osteoporosis and fractures. This is consistent with clinical and histological studies, suggesting that osteoblast differentiation is suppressed in diabetes. Osmoadaptation in eukaryotic cells involves the upregulation of PKC, p38 and AP-1 activities to induce expression of genes involved in cell volume regulation. If elevated, these signaling pathways can also have phenotypic consequences to developing and mature osteoblasts. We and others have demonstrated that AP-I family members influence osteoblast differentiation and gene expression. Correspondingly, long-term exposure of osteoblasts or calvaria to hyperosmotic conditions causes decreased mineralization. To test our hypothesis, the following Aims will be addressed: (1) determine levels of AP-1 and CRE activity and their importance in osteoblast responsiveness to hyperglycemia (HG) & hyperosmolarity (HO); (2) identify and test the roles of selected protein kinases in AP-1 and CRE activation by HG and HO; and (3) determine and test the roles of AP-1 and CRE activation in HG and HO modulation of target genes and bone phenotype in vivo. The first two Aims of this proposal will focus on examining molecular targets and signaling pathways modulated in response to hyperglycemia and hyperosmolarity. We hypothesize that the response to both conditions is mechanistically linked and will test if genes associated with an osmoadaptation are induced under HG conditions. Aim 3 will test if our cellular and molecular models are consistent with in vivo responses to HG. In each Aim, we will test if we can block osteoblast osmoadaptation and restore osteoblast phenotype. Taken together, the goals of this study are to determine the effects of osmoadaptation on osteoblast phenotype and to identify those components of this mechanism that impede osteoblast differentiation, both in vitro and in vivo. Our results will suggest targets for ameliorating the effects of type I diabetes on bone. [unreadable] [unreadable]

INTELLECTUAL MERIT: Hydroxyapatite (HA) is a calcium phosphate mineral resembling the natural mineral apatite phase of bone and widely used to fabricate scaffolds to support bone cell growth in implanted bone grafts. This proposal explores the hypothesis that smaller grain sizes and microcracking caused by thermal expansion anisotropy (TEA) in HA materials designed for use as bone grafts (1) increase the number and maturation of osteoblast (bone forming) cells adhering to the graft, (2) foster deposition of mineralized tissue and microcrack healing, and (3) promote improved mechanical strength and performance of the graft. These hypotheses are consistent with published and preliminary laboratory findings that definitively identified microcracking in HA, postulated microcracking as a toughening mechanism for brittle ceramics such as HA, demonstrated that HA enhances osteoblast function, and showed increased osteoblast proliferation on nanograined HA. The project has three objectives: (1) To determine the extent of microcrack damage in HA and correlate the microcracking with osteoblast attachment and maturation. (2) To determine the localized occurrence of microcrack healing by osteoblasts on HA and correlate such healing with measurable changes in global and local material properties such as the elastic modulus. (3) To determine the role of grain boundaries on osteoblast attachment and maturation and correlate osteoblast activity with grain size.

BROADER IMPACTS: Approximately 500,000 bone grafts are performed in the United States annually. Common sources for bone graft material, both autografts and allografts, are highly limited by availability, quantity, poor mechanical properties, risk of disease transmission, and cost. There is an urgent need for a biocompatible synthetic engineered bone graft material with adequate mechanical integrity and enhanced healing/bone integration properties. The expectation is that the neobone bioceramic constructs developed in this project, because of their improved morphology and cellular activity, will be a viable synthetic alternative to all currently available graft materials for repair of damaged bone tissue. The project will support two graduate students throughout their doctoral training. The PIs have routinely supported undergraduate research workers on their projects, many of whom have been coauthors on journal articles and conference presentations. Members of the research team will participate each summer in the MSU Summer High School Engineering Institute for Detroit-area students, who include many women and minority participants.

DESCRIPTION (provided by applicant): Over 40 million Americans (14 million of which are men) of all ages are afflicted with low bone density and its associated increased risk of fractures. While aging is a major cause of osteoporosis, diseases (such as type I diabetes, cystic fibrosis, asthma, inflammatory bowel disease, and arthritis), inactivity, poor dietary choices, and certain medications can cause bone loss at any age. Recent studies demonstrate that bone communications with the intestine. Potential mechanisms include signaling to bone through gastrointestinal (GI) hormones and calcium absorption. Recently, we found another interaction: GI inflammation leads to increased bone inflammation, which is associated with suppressed bone formation and bone loss in mice. This suggests that therapies which improve overall intestinal health have the potential to benefit bone health. Identifying natural products that could help individuals attain their maximum bone mass and suppress bone loss would be of great preventative and therapeutic value. Probiotics, microorganisms that provide a health benefit to the host when ingested, are used to treat a wide variety of ailments ranging from diarrhea to allergies. These microorganisms may boost the immune system, reduce colonization of harmful bacteria in the intestine, destroy certain bacterial toxins, decrease intestinal inflammation and help with digestion and mineral absorption. To address the effects probiotics on bone, we administered an anti- inflammatory probiotic strain, Lactobacillus reuteri ATCC PTA 6475, to adult mice and measured their bone densities. Fascinatingly, we found that L. reuteri treatment decreased TNF levels in the ileum and increased bone volume by greater than 45% in healthy male but not female mice. Based on our data we hypothesize that ingestion of L. reuteri increases bone density in a gender dependent manner through suppression of intestinal inflammation and upregulation of bone formation. We further hypothesize that sex hormones and changes in other intestinal signals (microbiota composition, hormones, and calcium status) contribute to the bone response. We propose to test our hypotheses by 1) Identifying the influence of gender on bone responses to L. reuteri treatment and 2) Identifying characteristics of probiotic L. reuteri and its effects on the gastrointestinal tract which are critical for enhancing bone mass. Few studies have examined probiotic effects on bone, and none have included an extensive analysis of bone, serum and GI parameters. Our studies will fill this gap in knowledge by identifying basic mechanisms relating the GI tract to bone health and will test the influences of gender in this linkage. Given the increasing use of probiotics in the United States and worldwide, our findings warrant further investigation into the effects of probiotic use on bone health in males and females. PUBLIC HEALTH RELEVANCE: Finding effective novel treatments for bone loss is a priority, since it is estimated that by 2020 more than 61 million men and women will have osteoporosis and its associated increase in fracture risk and potential negative effects on metabolism and insulin secretion. We have identified a novel way of increasing bone mass by use of a probiotic bacterium that attenuates intestinal inflammation. If proven effective, it could lead to a significant improvement in the health and well being of people with decreased bone density.

DESCRIPTION (provided by applicant): Osteoporosis and osteopenia affect over 200 million people worldwide and result in $14 billion in health care costs annually in the US alone. With an increasing aging population it is estimated that the 67 million Americans will be afflicted by low bone mass by the year 2020. Post-menopausal women are by far the largest population that suffers from osteoporosis. Current treatments to prevent bone loss predominantly utilize pharmacologic agents that target osteoclast activity, but their long-term use can have unwanted side effects including increased risk of unusual fractures. Thus physicians and their patients are looking for novel prevention and treatment options. Available data support a role for pro-inflammatory cells and cytokines as therapeutic targets because of their role in mediating osteoclast maturation and activity during menopause. Intestine-bone signaling is demonstrated to contribute to the regulation of bone density, yet very little is known about how menopause impacts the intestine and the role that this could play in bone loss. We have identified, for the first time that estrogen deficiency (induced by ovariectomy, ovx) leads to intestinal inflammation and causes dysbiosis of the intestinal microbiota in mice. Most importantly, oral administration of a human derived probiotic, Lactobacillus reuteri ATCC PTA 6475, inhibits ovx induced intestinal changes, bone marrow T-cell changes and prevents bone loss in ovx mice. Based on our novel findings, we hypothesize that L. reuteri prevents ovx induced bone loss by reducing gut and bone inflammation. The following aims are proposed to elucidate the role of gut-bone interactions in ovx induced bone loss as well as the mechanisms of L. reuteri mediated microbiota changes that prevent osteoporosis. AIM 1. Investigate the role of the intestinal microbiota in ovx induced bone loss. AIM 2. Identify the host mechanisms by which L. reuteri 6475 prevents ovx induced bone inflammation and bone loss. AIM 3. Establish the mechanistic basis of L. reuteri 6475 prevention of osteoporosis in ovx. Outcomes of our studies could provide a paradigm shift in understanding the role of gut-bone signaling axis in menopause and the role of estrogen in shaping our intestinal microbiota. Our findings will also provide the foundation for future clinical studies to determine the effectiveness of probiotics to maximize bone health in post-menopausal women.

DESCRIPTION (provided by applicant): Reduced bone formation and osteoporosis are serious complications of type I diabetes (T1D) that predispose diabetic patients to increased fracture risk and reduced quality of life. Therefore, developing therapeutic strategies to target T1D bone loss is a critical need for diabetic patients. We have identified that oral administration of the probiotic L. reuteri strikingly prevents T1D femur and vertebral bone loss in two distinct T1D models of mice. Because probiotics, ingested bacteria that benefit health, modulate the gastrointestinal (GI) system, their beneficial effect on bone formation is intriguing and significat. Recent studies recognize the relationship between the GI system and bone as a critical factor in the regulation of bone density. In fact, intestinal inflammation and the gut microbiota have both been acknowledged as crucial players in the physiology of gut-bone axis. Given their beneficial effect on the GI system, probiotics are increasingly used to treat diseases such as colic and inflammatory bowel disease (IBD). The long-term goal of our research is to test the potential use of a probiotic therapeutic for treating T1D bone loss in human patients. The objective of this proposal is to further understand the mechanistic basis by which L. reuteri modifies gut-bone axis and prevents bone loss. Our data indicate that LR has beneficial effects in the gut including decreased inflammation, enhancement of barrier function, and microbial community composition changes. These changes appear to translate into improved bone health in mice. Our data further demonstrate that L. reuteri reverses T1D-suppression of skeletal Wnt10b, a critical enhancer of osteoblast lineage, differentiation and bone formation. Interestingly, Wnt10b expression and osteoblast viability are critically regulated by bone marrow-derived TNF? in T1D. Together our pilot data implicates gut microenvironment and bone TNF?-Wnt10b axis as potential mechanistic targets of LR for preventing T1D bone loss. Based on our novel findings we hypothesize that LR treatment prevents T1D bone loss through its actions on the gut and/or bone microenvironment by preventing intestinal inflammation and osteoblast death. We will test this hypothesis by 1) Identifying the mechanistic basis by which LR regulates the gut environment to prevent bone loss in T1D male mice and 2) Determining the host mechanisms by which LR treatment modulates bone microenvironment and prevents T1D-induced bone inflammation and bone loss. The bone anabolic properties of L. reuteri and its impeccable safety record make it a novel bio- therapeutic option with minimal side effects to reduce osteoporosis and fracture risk in T1D patients. Our studies could provide a paradigm shift in understanding the role of gut-bone signaling axis in T1D.