R. Nicole Howie

Inkjet-based biopatterning of SDF-1β augments BMP-2-induced repair of critical size calvarial bone defects in mice

BACKGROUND A major problem in craniofacial surgery is non-healing bone defects. Autologous reconstruction remains the standard of care for these cases. Bone morphogenetic protein-2 (BMP-2) therapy has proven its clinical utility, although non-targeted adverse events occur due to the high milligram-level doses used. Ongoing efforts explore the use of different growth factors, cytokines, or chemokines, as well as co-therapy to augment healing. METHODS Here we utilize inkjet-based biopatterning to load acellular DermaMatrix delivery matrices with nanogram-level doses of BMP-2, stromal cell-derived factor-1β (SDF-1β), transforming growth factor-β1 (TGF-β1), or co-therapies thereof. We tested the hypothesis that bioprinted SDF-1β co-delivery enhances BMP-2 and TGF-β1-driven osteogenesis both in-vitro and in-vivo using a mouse calvarial critical size defect (CSD) model. RESULTS Our data showed that BMP-2 bioprinted in low-doses induced significant new bone formation by four weeks post-operation. TGF-β1 was less effective compared to BMP-2, and SDF-1β therapy did not enhance osteogenesis above control levels. However, co-delivery of BMP-2+SDF-1β was shown to augment BMP-2-induced bone formation compared to BMP-2 alone. In contrast, co-delivery of TGF-β1+SDF-1β decreased bone healing compared to TGF-β1 alone. This was further confirmed in vitro by osteogenic differentiation studies using MC3T3-E1 pre-osteoblasts. CONCLUSIONS Our data indicates that sustained release delivery of a low-dose growth factor therapy using biopatterning technology can aid in healing CSD injuries. SDF-1β augments the ability for BMP-2 to drive healing, a result confirmed in vivo and in vitro; however, because SDF-1β is detrimental to TGF-β1-driven osteogenesis, its effect on osteogenesis is not universal.

A Model for Osteonecrosis of the Jaw with Zoledronate Treatment following Repeated Major Trauma

This study aims to develop a reproducible rat model for post-traumatic bisphosphonate-related osteonecrosis of the jaw (BRONJ). In our previous studies using dental extraction as an inducing factor, only 30% - 60% of zoledronate-treated animals fulfilled the definition of clinical BRONJ. We modified the zoledronate regimen and introduced repeated surgical extraction to illicit quantifiable BRONJ in all animals. Eighty retired-breeder female Sprague-Dawley rats were divided between the treatment (IV zoledronate; 80 μg/kg/week for 13 weeks) and control (saline) groups. On week 13, the left mandibular first molar was surgically extracted, followed by the second molar a week later. Animals were euthanized at 1-week, 2-weeks, and 8-weeks following extraction. The occurrence and severity of BRONJ were scored in each animal based on gross and MicroCT analysis. Parameters of bone formation and osteoclast functions at the extraction site were compared between groups. All zoledronate-treated animals developed a severe case of BRONJ that fulfilled the clinical definition of the condition in humans. Osteoclast attachment continued to be defective eight weeks after stopping the treatment. There were no signs of kidney or liver toxicity. Our data confirmed that repeated surgical extraction (major trauma) by itself consistently precipitated massive bone necrosis in ZA-treated animals, eliminating the need to induce pre-existing infection or comorbidity. These results will be the basis for further studies examining the in-vivo pathogenesis and prevention of BRONJ.

Selective serotonin reuptake inhibitor exposure alters osteoblast gene expression and craniofacial development in mice

BACKGROUND Selective serotonin reuptake inhibitor (SSRI) use in pregnancy has been linked to craniofacial birth defects. Little is known about the effects of serotonin or SSRIs on craniofacial development. Here, we provide evidence that citalopram (SSRI) alters the osteogenic profile of murine calvarial cells and leads to craniofacial dysmorphology. METHODS We used mouse calvarial pre-osteoblast cells (MC3T3-E1) to study the biochemical profile (microarray and quantitative reverse transcription polymerase chain reactions) after treatment with a titrated dose of citalopram. We used C57BL-6 wild-type breeders to produce litters treated with a clinical dose of citalopram during the third trimester of pregnancy. We used micro-computed tomography and morphometric measures to determine effects on craniofacial development. RESULTS Controls included untreated cells and age matched untreated litters. We observed decreases in proliferation and increases in alkaline phosphatase activity after citalopram exposure. We confirmed altered expression of genes linked to osteogenesis including Ocn and significant increase in expression of Alp after 7 days of treatment. Our data suggest altered expression of several genes related to craniofacial development (Fgf2, Fgfr2, Tgfβr2 Irs1, Igf1) and statistically significant changes in expression for (Col2a1, Gdf6, Hmox1, and Notch1). We also observed changes in regulation of the serotonin pathway (Sert, Tph1, Tph2, Htr2a, Lrp5) after treatment with citalopram. After in utero exposure to citalopram, mice displayed shorter narrow snouts, more globular skulls and several craniofacial anomalies. CONCLUSION Our results provide confirmatory evidence that citalopram exposure is associated with cellular and morphological alterations of the craniofacial complex, which may have important implications for use during pregnancy.

Craniofacial shape variation in Twist1+/- mutant mice

Craniosynostosis (CS) is a relatively common birth defect resulting from the premature fusion of one or more cranial sutures. Human genetic studies have identified several genes in association with CS. One such gene that has been implicated in both syndromic (Saethre–Chotzen syndrome) and nonsyndromic forms of CS in humans is TWIST1. In this study, a heterozygous Twist1 knock out (Twist1+/−) mouse model was used to study the craniofacial shape changes associated with the partial loss of function. A geometric morphometric approach was used to analyze landmark data derived from microcomputed tomography scans to compare craniofacial shape between 17 Twist1+/− mice and 26 of their Twist1+/+ (wild type) littermate controls at 15 days of age. The results show that despite the purported wide variation in synostotic severity, Twist1+/− mice have a consistent pattern of craniofacial dysmorphology affecting all major regions of the skull. Similar to Saethre–Chotzen, the calvarium is acrocephalic and wide with an overall brachycephalic shape. Mutant mice also exhibited a shortened cranial base and a wider and shorted face, consistent with coronal CS associated phenotypes. The results suggest that these differences are at least partially the direct result of the Twist1 haploinsufficiency on the developing craniofacial skeleton. This study provides a quantitative phenotype complement to the developmental and molecular genetic research previously done on Twist1. These results can be used to generate further hypotheses about the effect of Twist1 and premature suture fusion on the entire craniofacial skeleton. Anat Rec, 297:826–833, 2014.

Mesenchymal stem cell expression of SDF-1β synergizes with BMP-2 to augment cell-mediated healing of critical-sized mouse calvarial defects.

Bone has the potential for spontaneous healing. This process, however, often fails in patients with comorbidities. Tissue engineering combining functional cells, biomaterials and osteoinductive cues may provide alternative treatment strategies. We have recently demonstrated that stromal cell‐derived factor‐1β (SDF‐1β) works in concert with bone morphogenetic protein‐2 (BMP‐2) to potentiate osteogenic differentiation of bone marrow‐derived mesenchymal stem/stromal cells (BMSCs). Here, we test the hypothesis that SDF‐1β overexpressed in Tet‐Off‐SDF‐1β BMSCs, delivered on acellular dermal matrix (ADM), synergistically augments BMP‐2‐induced healing of critical‐sized mouse calvarial defects. BMSC therapies alone showed limited bone healing, which was increased with co‐delivery of BMP‐2. This was further enhanced in Tet‐Off‐SDF‐1β BMSCs + BMP‐2. Only limited BMSC retention on ADM constructs was observed after 4 weeks in vivo, which was increased with BMP‐2 co‐delivery. In vitro cell proliferation studies showed that supplementing BMP‐2 to Tet‐Off BMSCs significantly increased the cell number during the first 24 h. Consequently, the increased cell numbers decreased the detectable BMP‐2 levels in the medium, but increased cell‐associated BMP‐2. The data suggest that SDF‐1β provides synergistic effects supporting BMP‐2‐induced, BMSC‐mediated bone formation and appears suitable for optimization of bone augmentation in combination therapy protocols. Copyright

Effects of In Utero thyroxine exposure on murine cranial suture growth

Large scale surveillance studies, case studies, as well as cohort studies have identified the influence of thyroid hormones on calvarial growth and development. Surveillance data suggests maternal thyroid disorders (hyperthyroidism, hypothyroidism with pharmacological replacement, and Maternal Graves Disease) are linked to as much as a 2.5 fold increased risk for craniosynostosis. Craniosynostosis is the premature fusion of one or more calvarial growth sites (sutures) prior to the completion of brain expansion. Thyroid hormones maintain proper bone mineral densities by interacting with growth hormone and aiding in the regulation of insulin like growth factors (IGFs). Disruption of this hormonal control of bone physiology may lead to altered bone dynamics thereby increasing the risk for craniosynostosis. In order to elucidate the effect of exogenous thyroxine exposure on cranial suture growth and morphology, wild type C57BL6 mouse litters were exposed to thyroxine in utero (control = no treatment; low ~167 ng per day; high ~667 ng per day). Thyroxine exposed mice demonstrated craniofacial dysmorphology (brachycranic). High dose exposed mice showed diminished area of the coronal and widening of the sagittal sutures indicative of premature fusion and compensatory growth. Presence of thyroid receptors was confirmed for the murine cranial suture and markers of proliferation and osteogenesis were increased in sutures from exposed mice. Increased Htra1 and Igf1 gene expression were found in sutures from high dose exposed individuals. Pathways related to the HTRA1/IGF axis, specifically Akt and Wnt, demonstrated evidence of increased activity. Overall our data suggest that maternal exogenous thyroxine exposure can drive calvarial growth alterations and altered suture morphology.

Effects of thyroxine exposure on the Twist 1 +/− phenotype: A test of gene–environment interaction modeling for craniosynostosis

BACKGROUND Craniosynostosis, the premature fusion of one or more of the cranial sutures, is estimated to occur in 1:1800 to 2500 births. Genetic murine models of craniosynostosis exist, but often imperfectly model human patients. Case, cohort, and surveillance studies have identified excess thyroid hormone as an agent that can either cause or exacerbate human cases of craniosynostosis. METHODS Here we investigate the influence of in utero and in vitro exogenous thyroid hormone exposure on a murine model of craniosynostosis, Twist 1 +/-. RESULTS By 15 days post-natal, there was evidence of coronal suture fusion in the Twist 1 +/- model, regardless of exposure. With the exception of craniofacial width, there were no significant effects of exposure; however, the Twist 1 +/- phenotype was significantly different from the wild-type control. Twist 1 +/- cranial suture cells did not respond to thyroxine treatment as measured by proliferation, osteogenic differentiation, and gene expression of osteogenic markers. However, treatment of these cells did result in modulation of thyroid associated gene expression. CONCLUSION Our findings suggest the phenotypic effects of the genetic mutation largely outweighed the effects of thyroxine exposure in the Twist 1 +/- model. These results highlight difficultly in experimentally modeling gene-environment interactions for craniosynostotic phenotypes. Birth Defects Research (Part A) 106:803-813, 2016.

Removal of pamidronate from bone in rats using systemic and local chelation.

OBJECTIVES Bisphosphonates become adsorbed on hydroxyapatite crystals in the bone matrix. In case of side-effects, stopping the treatment would not affect the bisphosphonates already deposited in bone. This study tests the feasibility of in-vivo targeted removal of bisphosphonates from bone using chelating agents. DESIGN 32 Sprague Dawley rats were given an injection of fluorescent pamidronate (OsteoSense EX; 0.16nmol/g). They were treated with either systemic (cadmium) or local [ethylenediaminetetraacetic (EDTA) or citric acid (CA)] chelating agents to induce the removal of the bisphosphonate from bone. We evaluated the decrease in fluorescence in the alveolar bone, femur, tibia, and vertebrae. We also analyzed the systemic effects of treatment. RESULTS Systemic chelation reduced the pamidronate signal universally. However, the maximum reduction was observed in the alveolar bone and femur (22% and 21%, p values 0.008 and 0.028, respectively). Systemic chelation did not impair calcium homeostasis. The chelation effect was not due to a systemic toxic effect on the liver or kidney. On the other hand local chelation at the extraction site significantly (p=0.011) decreased the pamidronate signal at bony surfaces of the socket. CONCLUSIONS Systemic and local chelating agents can remove bisphosphonate from bone. This study establishes a new concept for the prevention of side effects of bisphosphonates during high-risk situations.

Thyroxine Exposure Effects on the Cranial Base

Thyroid hormone is important for skull bone growth, which primarily occurs at the cranial sutures and synchondroses. Thyroid hormones regulate metabolism and act in all stages of cartilage and bone development and maintenance by interacting with growth hormone and regulating insulin-like growth factor. Aberrant thyroid hormone levels and exposure during development are exogenous factors that may exacerbate susceptibility to craniofacial abnormalities potentially through changes in growth at the synchondroses of the cranial base. To elucidate the direct effect of in utero therapeutic thyroxine exposure on the synchondroses in developing mice, we provided scaled doses of the thyroid replacement drug, levothyroxine, in drinking water to pregnant C57BL6 wild-type dams. The skulls of resulting pups were subjected to micro-computed tomography analysis revealing less bone volume relative to tissue volume in the synchondroses of mouse pups exposed in utero to levothyroxine. Histological assessment of the cranial base area indicated more active synchondroses as measured by metabolic factors including Igf1. The cranial base of the pups exposed to high levels of levothyroxine also contained more collagen fiber matrix and an increase in markers of bone formation. Such changes due to exposure to exogenous thyroid hormone may drive overall morphological changes. Thus, excess thyroid hormone exposure to the fetus during pregnancy may lead to altered craniofacial growth and increased risk of anomalies in offspring.

Testing a novel nanofibre scaffold for utility in bone tissue regeneration

Many variables serve to alter the process of bone remodelling and diminish regeneration including the size and nature of the wound bed and health status of the individual. To overcome these inhibitory factors, tissue‐engineered osteoconductive scaffolds paired with various growth factors have been utilized clinically. However, many limitations still remain, for example, bone morphogenetic protein 2 (BMP2) can lead to rampant inflammation, ectopic bone formation, and graft failure. Here, we studied the ability for a nanofiber scaffold (Talymed) to accelerate BMP2 growth factor‐induced bone healing compared with the traditional absorbable collagen sponge (ACS) delivery system. One hundred fifty‐five adult wild type mice were arranged in 16 groups by time, 4 and 8 weeks, and treatment, ACS or Talymed, loaded with control, low, medium, or high dosages of BMP2. Skulls were subjected to microCT, biomechanical, and histological analysis to assess bone regeneration. The use of Talymed within the defect site was found to decrease the bone volume, bone formation rate, and alkaline phosphatase activity compared with ACS/BMP2 combinations. Interestingly, though Talymed regenerated less bone, the regenerate was found to have a greater hardness value than that of bone within the ACS groups. However, the difference in bone hardness between scaffolds was not detectable by 8 weeks. Based on these results, we found that the nanofiber scaffold generated a better quality of bone regenerate at 4 weeks but, due to the lack of overall bone formation and the inhibition of normal remodelling processes, was not as efficacious as the current clinical standard ACS/BMP2 therapy.