Megan E. Oest

Coating of Biomaterial Scaffolds with the Collagen-Mimetic Peptide GFOGER for Bone Defect Repair

Healing large bone defects and non-unions remains a significant clinical problem. Current treatments, consisting of auto and allografts, are limited by donor supply and morbidity, insufficient bioactivity and risk of infection. Biotherapeutics, including cells, genes and proteins, represent promising alternative therapies, but these strategies are limited by technical roadblocks to biotherapeutic delivery, cell sourcing, high cost, and regulatory hurdles. In the present study, the collagen-mimetic peptide, GFOGER, was used to coat synthetic PCL scaffolds to promote bone formation in critically-sized segmental defects in rats. GFOGER is a synthetic triple helical peptide that binds to the alpha(2)beta(1) integrin receptor involved in osteogenesis. GFOGER coatings passively adsorbed onto polymeric scaffolds, in the absence of exogenous cells or growth factors, significantly accelerated and increased bone formation in non-healing femoral defects compared to uncoated scaffolds and empty defects. Despite differences in bone volume, no differences in torsional strength were detected after 12 weeks, indicating that bone mass but not bone quality was improved in this model. This work demonstrates a simple, cell/growth factor-free strategy to promote bone formation in challenging, non-healing bone defects. This biomaterial coating strategy represents a cost-effective and facile approach, translatable into a robust clinical therapy for musculoskeletal applications.

3D imaging of tissue integration with porous biomaterials

Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.

Intrauterine exposure to high saturated fat diet elevates risk of adult-onset chronic diseases in C57BL/6 mice.

BACKGROUND The developmental environment is thought to determine, in part, lifelong metabolic parameters and risk of adult disease. Effects of maternal malnutrition on fetal growth have been studied extensively, and the role of poor prenatal diet in elevating lifelong risk of cardiovascular and metabolic disease has been well characterized (www.thebarkertheory.com). However, the contribution of gestational high saturated fat diet (HFD) to adult-onset metabolic disease and skeletal dysfunction has only recently been recognized, and as such is incompletely understood. METHODS The present study evaluates the pathophysiologic mechanisms linking gestational HFD (approximating the macronutrient content of fast food) and elevated oxidative stress (OS) to adult-onset skeletal, cardiovascular, and metabolic dysfunction. RESULTS Results of this study demonstrate that adult offspring of dams fed HFD during pregnancy exhibited adult hyperglycemia, insulin resistance, obesity, and hypertension, despite being fed healthy standard rodent chow throughout postnatal life. These offspring also showed significantly lower femoral epiphyseal average bone mineral density (ABMD) at 6 months of age, and dysregulation of distal femoral trabecular architecture at 12 months of age, characteristic of osteoporosis. Incidence of these adult-onset adverse skeletal and metabolic effects was reduced by supplementing the pregnant dam with the antioxidant (quercetin, Q) during pregnancy. CONCLUSIONS Collectively, these data suggest that offspring of dams who consume a diet rich in saturated fats during pregnancy are at increased risk of adult-onset chronic disease. Additionally, these chronic diseases were determined to be in-part OS-mediated, and preventable by increasing a prenatal dietary antioxidant; this knowledge offers both a putative mechanism of disease pathogenesis and suggests a potential preventive strategy.

Analyzing bone, blood vessels, and biomaterials with microcomputed tomography

Quantitative tools such as micro-CT are needed for tissue engineering to evolve beyond a qualitative, observational field and accelerate the clinical realization of regenerative technologies. As faster, higher-resolution micro-CT systems become available for both in vitro and in vivo studies and the development of improved contrast agents allows micro-CT imaging to be extended to nonmineralized tissues, additional novel applications related to tissue engineering are sure to emerge.

Protein antimicrobial barriers to bacterial adhesion: in vitro and in vivo evaluation of nisin-treated implantable materials

Abstract A novel approach to controlling unwanted microbial adhesion in clinical environments is to inhibit the initial attachment of bacteria, rather than trying to remove them once they have adhered. Previous investigations have established that antimicrobial peptides such as nisin can adsorb to surfaces and still retain sufficient activity to inhibit pathogenic bacteria. We examined techniques of application of nisin in vitro to elucidate those most effective and practical for use on biomedical implants in vivo. Nisin adsorbed quickly on Polyvinyl chloride (PVC) suction catheter tubing, with only a slight gain in nisin activity as contact times increased from 10 s to 8 h. The activity of nisin adsorbed on PVC suction catheter tubing increased as solution concentrations increased from 0.01 to 2.0 mg/ml, and it decreased with aging in protein-free phosphate buffer for 48 h and with drying for up to 2 months. When exposed to three species of Gram-positive bacteria, nisin-treated PVC tubing demonstrated an ability to inhibit bacterial growth, while bacteria grew unchecked on catheter material that was untreated. We then examined the ability of nisin to retain its activity in vivo when placed on implants in blood vessels or the upper airway and whether nisin causes tissue reactions greater than untreated implants placed in sheep and ponies. Freshly prepared nisin was applied to Teflon® FEP intravenous catheters and to PVC tracheotomy tubes at the time of placement, using a concentration of 1.0 mg/ml and 10-s contact time. Tissue reactions in response to nisin adsorbed on intravenous catheters or tracheotomy tubes did not occur in sheep or ponies, respectively. Nisin activity was retained for more than 5 h but less than 1 week on intravenous catheters placed in the jugular veins of sheep, and the veins with short-term catheters showed fewer and less severe histologic abnormalities compared with controls, indicating a possible protective effect on vascular endothelium. Nisin activity was retained on PVC tracheotomy tubes maintained for 1–2 h in ponies, but not on tubes in place for 24 h. As the first preclinical trial of nisin-treated implantable materials, this study represents an important first step for developing the potentially broad use of protein antimicrobial films on implantable medical devices.

Cyclic Mechanical Compression Increases Mineralization of Cell-Seeded Polymer Scaffolds In Vivo

Despite considerable documentation of the ability of normal bone to adapt to its mechanical environment, very little is known about the response of bone grafts or their substitutes to mechanical loading even though many bone defects are located in load-bearing sites. The goal of this research was to quantify the effects of controlled in vivo mechanical stimulation on the mineralization of a tissue-engineered bone replacement and identify the tissue level stresses and strains associated with the applied loading. A novel subcutaneous implant system was designed capable of intermittent cyclic compression of tissue-engineered constructs in vivo. Mesenchymal stem cell-seeded polymeric scaffolds with 8 weeks of in vitro preculture were placed within the loading system and implanted subcutaneously in male Fisher rats. Constructs were subjected to 2 weeks of loading (3 treatments per week for 30 min each, 13.3 N at 1 Hz) and harvested after 6 weeks of in vivo growth for histological examination and quantification of mineral content. Mineralization significantly increased by approximately threefold in the loaded constructs. The finite element method was used to predict tissue level stresses and strains within the construct resulting from the applied in vivo load. The largest principal strains in the polymer were distributed about a modal value of -0.24% with strains in the interstitial space being about five times greater. Von Mises stresses in the polymer were distributed about a modal value of 1.6 MPa, while stresses in the interstitial tissue were about three orders of magnitude smaller. This research demonstrates the ability of controlled in vivo mechanical stimulation to enhance mineralized matrix production on a polymeric scaffold seeded with osteogenic cells and suggests that interactions with the local mechanical environment should be considered in the design of constructs for functional bone repair.

Raman spectroscopy demonstrates prolonged alteration of bone chemical composition following extremity localized irradiation

INTRODUCTION Radiotherapy to the appendicular skeleton can cause an increased risk of developing catastrophic fractures with delayed bone healing or non-union, and may subsequently require multiple procedures and amputation. Biomechanical studies suggest that irradiated bone is more brittle, but the cause is unclear and cannot be explained by changes to bone structure or quantity, suggesting that there are crucial changes in irradiated bone material properties. Raman spectroscopy provides a means to assess the chemical properties of the mineral and matrix constituents of bone, which could help explain post-radiation embrittlement. In this study we use a murine tibial model with focal irradiation and perform Raman spectroscopy to test the hypothesis that changes in bone chemistry following irradiation is consistent with reduced bone quality and persists in the long term after irradiation. METHODS Female BALB/F mice aged 12weeks were subjected to unilateral, localized hindlimb irradiation in 4 daily 5Gy fractions (4×5Gy) totaling 20Gy, and were euthanized at 1, 4, 8, 12, and 26weeks post-irradiation (n=6/group). The irradiated (right) and non-irradiated contralateral control (left) tibiae were explanted and assessed by non-polarized and polarized Raman spectroscopy over the proximal cortical bone surface. Raman parameters used included the mineral/matrix ratio, mineral crystallinity, carbonate/phosphate ratio, collagen cross-link ratio, and depolarization ratio. RESULTS Significantly increased collagen cross-link ratio and decreased depolarization ratio of matrix were evident at 1week after irradiation and this persisted through 26weeks. A similar significant decrease was observed for depolarization ratio of mineral at all time points except 8 and 26weeks. At 4weeks after irradiation there was a significantly increased mineral/matrix ratio, increased mineral crystallinity, and decreased carbonate/phosphate ratio compared to controls. However, at 12weeks after irradiation these parameters had moved in the opposite direction, resulting in a significantly decreased mineral/matrix ratio, decreased crystallinity and increased carbonate/phosphate ratio compared to controls. At 26weeks, mineral/matrix, crystallinity and carbonate/phosphate ratios had returned to normal. DISCUSSION In this mouse model, Raman spectroscopy reports both bone mineral and collagen cross-link radiation-induced abnormalities that are evident as early as one week after irradiation and persists for 26weeks. The picture is one of extensive damage, after which there is an attempt at remodeling. We hypothesize that pathological cross-links formed by radiation damage to collagen are poorly resorbed during the altered remodeling process, so that new tissue is formed on a defective scaffold, resulting in increased bone brittleness.

Micro-CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear-staining methods.

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear-staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro-CT) imaging is a versatile, well-documented tool traditionally used to generate high-resolution 3-D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro-CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro-CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro-CT measurement of fetal limb lengths correlates well with traditional clear-staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro-CT and 10.82% for clear-staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro-CT technique to generate 3-D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear-staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well.

Gestational high saturated fat diet alters C57BL/6 mouse perinatal skeletal formation

BACKGROUND Our present work joins growing evidence that gestational environment (maternal nutrition, health, and chemical exposures) strongly influences prenatal development (www.thebarkertheory.org). The present study suggests that maternal consumption of a diet high in saturated fats (HFD), which approximates the macronutrient content of fast food, impairs perinatal skeletal development. METHODS In this study, administration of HFD (32% saturated fat) for one month prior to conception and throughout gestation in C57BL/6J mice was associated with a marked reduction in late-gestation fetal skeletal developmental delay that included shorter long bone lengths, decreased average bone mineral density (ABMD; 20%), lower total bone volume (TBV; 45%), and shorter crown-to-rump length (C-R; 12%), as compared to controls. RESULTS A putative mechanism linking prenatal HFD to dysregulated fetal osteogenesis is HFD-induced oxidative stress (OS), which has been shown in our laboratory to cause placental labyrinthine vascular damage and impaired fetal signaling pathways associated with osteogenesis (Liang et al., unpublished data). CONCLUSIONS The theory of HFD-associated, OS-mediated placental damage and skeletal pathogenesis was supported by demonstrating a protective effect of the dietary antioxidant quercetin (Q) against HFD-associated fetal skeletal developmental delay. Improved understanding of the role of HFD and elevated OS in fetal skeletal development will help to more completely elucidate the importance of the prenatal environment to fetal formation, and will be applied to better understand the contribution of the fetal environment to long-term risk of adult-onset disease.

Zoledronic Acid Prevents Loss of Trabecular Bone after Focal Irradiation in Mice

Radiation therapy for soft tissue sarcomas and metastatic disease can adversely affect bone, leading to late-onset fragility fractures. Adjunct administration of bisphosphonates has been postulated as means of minimizing these adverse effects. Using a murine model of focal hindlimb irradiation, we examined the potential for zoledronic acid treatment to minimize the deleterious effects of localized radiotherapy (RTx) on bone. Mice received a single, unilateral hindlimb exposure of 20 Gy. Beginning 4 days prior to irradiation, and at 1, 2 and 3 weeks post-irradiation, animals were treated with zoledronic acid or saline/vehicle injections. Areal bone mineral density was assessed at 4 days, and 2, 4 and 12 weeks post-irradiation by dual-energy X-ray absorptiometry (DXA). Micro-computed tomography and axial compression testing were used to quantify changes in morphological and mechanical properties of femurs at 4 and 12 weeks post-irradiation. Radiation had differential effects on cortical and trabecular bone, increasing cortical bone mineral content (BMC), cortical bone volume (BV) and trabecular separation (Tb.Sp) while decreasing trabecular number (Tb.N) by 12 weeks after localized radiotherapy. Administration of zoledronic acid increased hindlimb areal bone mineral density in both the presence and absence of radiotherapy, increased cortical bone mineral content and bone volume, increased trabecular bone volume (BV/TV), increased trabecular number, increased trabecular thickness (Tb.Th), and decreased trabecular separation compared to irradiated and vehicle control femurs. Despite these improvements in morphology with zoledronic acid, no biomechanical advantage was observed. Further work is needed to define the role of bisphosphonates in prevention of post-irradiation fragility fractures.