Mitigation of BMP-induced inflammation in craniofacial bone regeneration and improvement of bone parameters by dietary hesperidin.

Based on anti-inflammatory and osteogenic properties of hesperidin (HE), we hypothesized its systemic administration could be a cost-effective method of improving BMP-induced bone regeneration. Sprague-Dawley rats were allocated into 4 groups (n = 10/group): a 5-mm critical-sized mandible defect + collagen scaffold or, scaffold + 1 µg of BMP2 with and without dietary HE at 100 mg/kg. HE was administered by oral gavage 4 weeks prior to surgeries until euthanasia at day 7 or 14 post-surgery. The healing tissue within the defect collected at day 7 was subjected to gene expression analysis. Mandibles harvested at day 14 were subjected to microcomputed tomography and histology. HE + BMP2-treated rats had a statistically significant decrease in expression of inflammatory genes compared to BMP2 alone. The high-dose BMP2 alone caused cystic-like regeneration with incomplete defect closure. HE + BMP2 showed virtually complete bone fusion. Collagen fibril birefringence pattern (red color) under polarized light indicated high organization in BMP2-induced newly formed bone (NFB) in HE-supplemented group (p < 0.05). Clear changes in osteocyte lacunae as well as a statistically significant increase in osteoclasts were found around NFB in HE-treated rats. A significant increase in trabecular volume and thickness, and trabecular and cortical density was found in femurs of HE-supplemented rats (p < 0.05). Our findings show, for the first time, that dietary HE has a remarkable modulatory role in the function of locally delivered high-dose BMP2 in bone regeneration possibly via control of inflammation, osteogenesis, changes in osteocyte and osteoclast function and collagen maturation in regenerated and native bone. In conclusion, HE had a significant skeletal bone sparing effect and the ability to provide a more effective BMP-induced craniofacial regeneration.

Systemic Dietary Hesperidin Modulation of Osteoclastogenesis, Bone Homeostasis and Periodontal Disease in Mice.

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (µCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using µCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo.

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.

Effect of Flavonoid Supplementation on Alveolar Bone Healing-A Randomized Pilot Trial.

We investigated the effects of two common dietary supplements on bone healing in dental extraction sockets in humans. In this randomized pilot trial, male subjects took Grape Seed Extract [GSE] or Grapefruit Extract [GFE] starting two weeks prior to dental extraction and maintained this regimen for sixty days after surgery. Extraction sockets were filled with a collagen plug. After 24 h, a socket sample was collected and processed for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and an 84-gene wound healing assay. Sixty days after tooth extraction, a core of newly formed bone was obtained prior to dental implant placement and processed for histology. qRT-PCR revealed that GFE led to a significant decrease in platelet-derived growth factor and interleukin (IL)1-ß compared to GSE, and a significant decrease in IL-6 and CXCL2 compared to control. GSE led to a significant increase in coagulation factor Von Willebrand and inflammatory marker IL1-ß compared to GFE. WISP1 and CXCL5 were upregulated in both groups. Overall, GFE showed a downregulation of inflammation and GSE led to a decrease in collagen density and increased osteoclasts. This pilot trial highlights the need for further investigation on the mechanism of action of such supplements on bone healing and oral health.

Developing Implantable Scaffolds to Enhance Neural Stem Cell Therapy for Post-Operative Glioblastoma.

Pre-clinical and clinical studies have shown that engineered tumoricidal neural stem cells (tNSCs) are a promising treatment strategy for the aggressive brain cancer glioblastoma (GBM). Yet, stabilizing human tNSCs within the surgical cavity following GBM resection is a significant challenge. As a critical step toward advancing engineered human NSC therapy for GBM, we used a preclinical variant of the clinically utilized NSC line HB1.F3.CD and mouse models of human GBM resection/recurrence to identify a polymeric scaffold capable of maximizing the transplant, persistence, and tumor kill of NSC therapy for post-surgical GBM. Using kinetic bioluminescence imaging, we found that tNSCs delivered into the mouse surgical cavity wall by direct injection persisted only 3 days. We found that delivery of tNSCs into the cavity on nanofibrous electrospun poly-l-lactic acid scaffolds extended tNSC persistence to 8 days. Modifications to fiber surface coating, diameter, and morphology of the scaffold failed to significantly extend tNSC persistence in the cavity. In contrast, tNSCs delivered into the post-operative cavity on gelatin matrices (GEMs) persisted 8-fold longer as compared to direct injection. GEMs remained permissive to tumor-tropic homing, as tNSCs migrated off the scaffolds and into invasive tumor foci both in vitro and in vivo. To mirror envisioned human brain tumor trials, we engineered tNSCs to express the prodrug/enzyme thymidine kinase (tNSCstk) and transplanted the therapeutic cells in the post-operative cavity of mice bearing resected orthotopic patient-derived GBM xenografts. Following administration of the prodrug ganciclovir, residual tumor volumes in mice receiving GEM/tNSCs were reduced by 10-fold at day 35, and median survival was extended from 31 to 46 days. Taken together, these data begin to define design parameters for effective scaffold/tNSC composites and suggest a new approach to maximizing the efficacy of tNSC therapy in human patient trials.

Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications.

UNLABELLED: The fabrication and characterization of novel high surface area hollow gilled fiber tissue engineering scaffolds via industrially relevant, scalable, repeatable, high speed, and economical nonwoven carding technology is described. Scaffolds were validated as tissue engineering scaffolds using human adipose derived stem cells (hASC) exposed to pulsatile fluid flow (PFF). The effects of fiber morphology on the proliferation and viability of hASC, as well as effects of varied magnitudes of shear stress applied via PFF on the expression of the early osteogenic gene marker runt related transcription factor 2 (RUNX2) were evaluated. Gilled fiber scaffolds led to a significant increase in proliferation of hASC after seven days in static culture, and exhibited fewer dead cells compared to pure PLA round fiber controls. Further, hASC-seeded scaffolds exposed to 3 and 6dyn/cm(2) resulted in significantly increased mRNA expression of RUNX2 after one hour of PFF in the absence of soluble osteogenic induction factors. This is the first study to describe a method for the fabrication of high surface area gilled fibers and scaffolds. The scalable manufacturing process and potential fabrication across multiple nonwoven and woven platforms makes them promising candidates for a variety of applications that require high surface area fibrous materials. STATEMENT OF SIGNIFICANCE: We report here for the first time the successful fabrication of novel high surface area gilled fiber scaffolds for tissue engineering applications. Gilled fibers led to a significant increase in proliferation of human adipose derived stem cells after one week in culture, and a greater number of viable cells compared to round fiber controls. Further, in the absence of osteogenic induction factors, gilled fibers led to significantly increased mRNA expression of an early marker for osteogenesis after exposure to pulsatile fluid flow. This is the first study to describe gilled fiber fabrication and their potential for tissue engineering applications. The repeatable, industrially scalable, and versatile fabrication process makes them promising candidates for a variety of scaffold-based tissue engineering applications.

Interconnected, microporous hollow fibers for tissue engineering: commercially relevant, industry standard scale-up manufacturing.

Significant progress has been achieved in the field of tissue engineering to create functional tissue using biomimetic three-dimensional scaffolds that support cell growth, proliferation, and extracellular matrix production. However, many of these constructs are severely limited by poor nutrient diffusion throughout the tissue-engineered construct, resulting in cell death and tissue necrosis at the core. Nutrient transport can be improved by creation and use of scaffolds with hollow and microporous fibers, significantly improving permeability and nutrient diffusion. The purpose of this review is to highlight current technological advances in the fabrication of hollow fibers with interconnected pores throughout the fiber walls, with specific emphasis on developing hollow porous nonwoven fabrics for use as tissue engineering constructs via industry standard processing technologies: Spunbond processing and polymer melt extrusion. We outline current methodologies to create hollow and microporous scaffolds with the aim of translating that knowledge to the production of such fibers into nonwoven tissue engineering scaffolds via spunbond technology, a commercially relevant and viable melt extrusion manufacturing approach that allows for facile scale-up.