The Application of Melatonin and Platelet-Rich Plasma in the Development of a Bioactive Calcium Aluminate Bone Regenerative Scaffold

Over 500,000 bone graft procedures are conducted annually within the United States. Autografts contribute to donor site complications and disease transmission with allografts has been described. Many ceramics are only osteoconductive and are brittle, limiting their clinical use. Thus, the objective of this study was to create a bone substitute with osteoinductive properties similar to natural bone using the ceramic biomaterial calcium aluminate (CA). Calcium aluminate materials are durable and remain moldable for an extended period of time at room temperature. Further, the surfaces of CA scaffolds can be modified with biological agents through simple chemical means to locally deliver agents directly to sites of injury. In order to enhance local bone regenerating characteristics of CA scaffolds, melatonin and platelet-rich plasma (PRP) were utilized for their known osteoinductive properties. Platelet-rich plasma enhances soft and hard tissue formation primarily through growth factor-mediated signaling pathways. Melatonin augments osteoblast differentiation and inhibits osteoclast-mediated bone resorption through receptor-dependent signaling and free radical scavenging activity, respectively. Thus, it was hypothesized that melatonin and/or PRP would provide osteoinductive properties to CA scaffolds to promote bone regeneration in a rodent model of critical-size calvaria defects. Modified CA scaffolds (CA-Mel) were produced by immobilizing melatonin to the CA surface through a covalent linkage. The biocompatibility of unmodified and modified CA scaffolds was initially tested in vitro and indicated that modified surfaces had a preference for the adhesion and proliferation of normal human osteoblasts versus NIH 3T3 fibroblasts. Moreover, the immobilization of melatonin to the CA surface may delay the differentiation of human adult mesenchymal stem cells (hAMSCs) and may have facilitated their migration across the CA surface. Two-month-old ovariectomized rats were randomized into implant groups receiving unmodified or modified scaffolds in the absence (CA and CA-Mel) or presence of PRP (CA+PRP and CA-Mel+PRP). Histological sections confirmed that both CA scaffold types were well-tolerated and provided evidence of tissue infiltration and scaffold biodegradation over time. Bone regeneration in animals was assessed by fluorochrome labeling at three and six months. While there was a lack of synergism between melatonin and PRP in the CA-Mel+PRP group, animals implanted with CA-Mel showed the greatest intensity and abundance of bone remodeling at both time points compared to all other groups. Radiographic data indicated a significant increase in the density of newly formed bone over time in all groups. The absence of a detectable decrease in density suggests that the modest biodegradation of CA scaffolds is balanced with processes of bone formation. Finally, both unmodified and modified CA scaffolds continued to provide a supportive surface for bone formation out to six months. Overall, results from this study suggest that CA scaffolds modified with melatonin may enhance bone remodeling activity in calvarial defects through hAMSC differentiation and recruitment and by preferentially supporting the viability and function of mature osteoblasts. This novel bioactive ceramic scaffold has the potential to change the dogma of bone grafting in fields like dentistry and reconstructive surgery. Continued optimization of this therapy is warranted and the attachment of other osteoinductive biomolecules is being considered

Morinda citrifolia (Noni) Juice Augments Mammary Gland Differentiation and Reduces Mammary Tumor Growth in Mice Expressing the Unactivated c-erbB2 Transgene

Morinda citrifolia (noni) is reported to have many beneficial properties, including on immune, inflammatory, quality of life, and cancer endpoints, but little is known about its ability to prevent or treat breast cancer. To test its anticancer potential, the effects of Tahitian Noni Juice (TNJ) on mammary carcinogenesis were examined in MMTV-neu transgenic mice. Mammary tumor latency, incidence, multiplicity, and metastatic incidence were unaffected by TNJ treatment, which suggests that it would not increase or decrease breast cancer risk in women taking TNJ for its other benefits. However, noni may be useful to enhance treatment responses in women with existing HER2/neu breast cancer since TNJ resulted in significant reductions in tumor weight and volume and in longer tumor doubling times in mice. Remarkably, its ability to inhibit the growth of this aggressive form of cancer occurred with the mouse equivalent of a recommended dose for humans (<3 oz/day). A 30-day treatment with TNJ also induced significant changes in mammary secondary ductule branching and lobuloalveolar development, serum progesterone levels, and estrous cycling. Additional studies investigating TNJ-induced tumor growth suppression and modified reproductive responses are needed to characterize its potential as a CAM therapy for women with and without HER2+ breast cancer

An Evaluation of the Efficacy of Morinda citrifolia (Noni) on the Development and Progression of Breast Cancer in the MMTV-neu Mouse Model

Seventy-nine percent of individuals consuming Morinda citrifolia (Noni), a popular, over-the-counter dietary supplement, have reported beneficial effects (of 25, 000 testimonials). A recent addition to the list of activities synonymous with Noni juice consumption includes its potential to prevent the development of cancer in some models. However, there is no in vivo data that suggests its potential efficacy against the development of human breast tumors. Therefore, investigation into the potential use of Noni as an adjuvant therapy for the prevention and treatment of breast cancer is warranted and formed the basis of this study. The effects of Tahitian Noni® Juice (TNJ) on the development and progression of mammary tumorigenesis were studied in the MMTV-neu mouse model. Investigating the effects of TNJ in the MMTV-neu model has several benefits, including the spontaneous development of mammary tumors and its relation to breast cancers that over-express HER2. Investigation into the mechanistic activity of TNJ in normal mammary glands (pre-tumor study) revealed that a thirty day treatment with 10% TNJ has the potential to down-regulate enzymes implicated in local hormone biosynthesis as well as the transcribed message for epidermal growth factor. Concomitantly, there was a 5-fold increase in β-casein expression, a known marker of mammary gland differentiation, in the mammary glands of treated mice. Mammary gland whole mount assessments supported significant changes in mammary gland architecture, including increases in secondary ductule branching (p 0.0001) and alveolobular development (p = 0.0015). Thirty day treatment with TNJ was also capable of causing a significant reduction in circulating levels of progesterone (p = 0.0316) and reduced the number of estrous cycles of treated mice (p = 0.0102). To investigate whether tumor outcomes were also modified, the long-term effects of 10% TNJ administration were examined. Mammary tumor latency, mammary tumor incidence, mammary tumor multiplicity, and metastatic incidence in the lungs were unaffected with 10% TNJ treatment compared to the control group. However, TNJ demonstrated potent growth inhibitory activity with significant reductions in mammary tumor weight and mammary tumor volume (p = 0.0074 and p = 0.0141, respectively), and a significant reduction in solid primary mammary tumor doubling time and growth rate for a specific window of tumor sizes (p = 0.0364 and p = 0.0427, respectively; 2500-4000 mm3). Finally, the long-term administration of TNJ did not result in hepato- and nephrotoxicity for the parameters analyzed in this study. The ability of TNJ to augment mammary gland differentiation may affect the perpetuation of events leading to continued tumor growth in the mammary tissue. Moreover, TNJ demonstrates the potential to reduce mammary tumor burden and growth rates in aggressive, hormone-independent cancers such as those over-expressing HER2. Ultimately, this may enhance the manageability and survivability of HER2 over-expressing breast cancer by allowing additional time for surgical or therapeutic intervention

Biomimetic tissue-engineered bone substitutes for maxillofacial and craniofacial repair : the potential of cell sheet technologies

Maxillofacial defects are complex lesions stemming from various etiologies: accidental, congenital, pathological, or surgical. A bone graft may be required when the normal regenerative capacity of the bone is exceeded or insufficient. Surgeons have many options available for bone grafting including the "gold standard" autologous bone graft. However, this approach is not without drawbacks such as the morbidity associated with harvesting bone from a donor site, pain, infection, or a poor quantity and quality of bone in some patient populations. This review discusses the various bone graft substitutes used for maxillofacial and craniofacial repair: allografts, xenografts, synthetic biomaterials, and tissue-engineered substitutes. A brief overview of bone tissue engineering evolution including the use of mesenchymal stem cells is exposed, highlighting the first clinical applications of adipose-derived stem/stromal cells in craniofacial reconstruction. The importance of prevascularization strategies for bone tissue engineering is also discussed, with an emphasis on recent work describing substitutes produced using cell sheet-based technologies, including the use of thermo-responsive plates and the self-assembly approach of tissue engineering. Indeed, considering their entirely cell-based design, these natural bone-like substitutes have the potential to closely mimic the osteogenicity, osteoconductivity, osteoinduction, and osseointegration properties of autogenous bone for maxillofacial and craniofacial reconstruction

The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes.

Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system

Human erythrocyte membrane engineering with NHS-pDMAA-Rh polymers.

<p>NHS-pDMAA-Rh polymers exhibit significant non-specific binding and initial high-density fluorescence quenching. Human RBCs were modified with 182 μM NHS-pDMAA-Rh or 98 μM HO-pDMAA-Rh for 30 minutes at 37°C. (<b>A</b>) Representative images of NHS-pDMAA-Rh-exposed hRBC for each designated time point. (<b>B</b>) Representative images of HO-pDMAA-Rh-exposed hRBC for each designated time point. Epifluorescent images were capture after washing on a Leica inverted microscope at 20X. Scale bars measure 100 μm. (<b>C</b>) Supernatant, cytosolic, and membrane fractions were collected at 0, 1, 4, and 8 hours. Polymer retention and internalization was assessed by monitoring the relative fluorescence of each fraction over time and calculated as the number of polymer molecules per hRBC using a standard curve. (<b>D</b>) The number of polymer molecules per nm² hRBC surface area was calculated using an average hRBC surface area of 140 μm². Images were background corrected and the brightness/contrast for each channel was balanced using Image J software. n = 3.</p

Effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on membrane stability.

<p>(<b>A</b>) Modification of hRBC with NHS-pDMAA-Cy3 polymers does not induce membrane destabilization as evidenced by the absence of PS-Annexin V signal under physiological conditions. Both unmodified and modified (100 μM polymer solution) hRBC were incubated in 1X PBS for one hour at 37°C. Annexin V-Alexa488 binding to externalized phosphatidylserine was used to monitor membrane destabilization and oxidative damage (green fluorescence). Cy3-modification demonstrated by red fluorescence. Epifluorescent images of PS-Annexin V and Cy3 channels captured under oil at 60x using an inverted Leica microscope. Individual channels were merged using Image J. Scale bars measure 50 μm. Images were background corrected and the brightness/contrast for each channel was balanced using Image J software. (<b>B</b>) Effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on membrane damage during oxidant exposure. Modification of hRBC with NHS-pDMAA-Cy3 polymers may protect against membrane damage under oxidizing conditions demonstrated by a potential mitigation of phosphatidylserine-Annexin V signal. Both unmodified and modified (100 μM polymer solution) hRBC were incubated in 0.2 mM CuSO₄/2.5 mM ascorbate solution for one hour at 37°C. Annexin V-Alexa488 binding to externalized phosphatidylserine was used to monitor membrane destabilization and oxidative damage (green fluorescence). Cy3-modification demonstrated by red fluorescence. Epifluorescent images of PS-Annexin V and Cy3 channels captured under oil at 60X using an inverted Leica microscope. Individual channels were merged using Image J. Scale bars measure 50 μm. Images were background corrected and the brightness/contrast for each channel was balanced using Image J software. (<b>C-F</b>). Time point analysis of the effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on membrane damage during oxidant exposure. Modification of hRBC with NHS-pDMAA-Cy3 polymers may protect against membrane damage under oxidizing conditions demonstrated by a potential mitigation of phosphatidylserine-Annexin V signal over time. To further investigate the temporal regulation of these events, both unmodified and modified (100 μM polymer solution) hRBC were incubated in 0.2 mM CuSO₄/2.5 mM ascorbate solution for 10 (<b>C</b>), 30 (<b>D</b>), 60 (<b>E</b>), or 120 (<b>F</b>) minutes at 37°C. Annexin V-Alexa488 binding to externalized phosphatidylserine was used to monitor membrane destabilization and oxidative damage (green fluorescence). Cy3-modification demonstrated by red fluorescence. Epifluorescent images of PS-Annexin V and Cy3 channels captured under oil at 60X using an inverted Leica microscope. Individual channels were merged using Image J. Scale bars measure 50 μm. Images were background corrected and the brightness/contrast for each channel was balanced using Image J software.</p

The effect of human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers on Band 3 aggregation.

<p>Human erythrocyte Band 3 protein is a major site of NHS-pDMAA-Cy3 modification supported by the absence of Band 3 aggregate formation in the presence of the protein cross-linker bis(sulfosuccinimidyl)suberate (BS₃). (<b>A</b>) Isolated hRBC membranes for each group were run on 12% TGX polyacrylamide gels and stained with Imperial protein stain. Gel image truncated after 37 kDa molecular weight marker to highlight larger molecular weight membrane proteins. (<b>B</b>) Histogram plots of band intensity versus migration distance were plotted for each experimental group and concentration (0 mM and 1 mM BS₃ concentrations shown). Dotted lines are used to represent peak intensity of specific protein bands in non-crosslinked hRBC membranes. Specifically, at the highest concentration of BS₃ (1 mM) there is a noticeable decrease in monomeric Band 3 protein band intensity as well as a modest increase in higher molecular weight proteins gathering at the top of the gel (e.g., in the region of spectrin and ankyrin proteins) for unmodified hRBCs versus those modified with NHS-pDMAA-Cy3 polymers. The arrow represents Band 3 aggregates.</p

Human erythrocyte membrane engineering with NHS-pDMAA-Cy3 polymers.

<p>NHS-pDMAA-Cy3 polymers are retained on hRBC membranes over a 24 hour period compared to HO-pDMAA-Cy3 polymers, indicating cell surface reactivity. Human RBCs were modified with 100 μM NHS-pDMAA-Cy3 or HO-pDMAA-Cy3 for 30 minutes at 37°C. (<b>A</b>) Representative images of NHS-pDMAA-Cy3-exposed hRBC for each designated time point. (<b>B</b>) Representative images of HO-pDMAA-Cy3-exposed hRBC for each designated time point. Epifluorescent images were capture after washing on a Leica inverted microscope at 40X. Scale bars measure 50 μm. (<b>C</b>) Supernatant, cytosolic, and membrane fractions were collected at 0, 1, 4, 8, and 24 hours. Polymer retention and internalization was assessed by monitoring the relative fluorescence of each fraction over time and calculated as the number of polymer molecules per hRBC using a standard curve. (<b>D</b>) The number of polymer molecules per nm² hRBC surface area was calculated using an average hRBC surface area of 140 μm². Images were background corrected and the brightness/contrast for each channel was balanced using Image J software. n = 3.</p

Schematic for the ATRP synthesis of a TEMPO-terminated poly(DMAA) polymer.

<p>The stable free-radical nitroxide, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO˙) was added to polymer chains to investigate its effects as a cytoprotective antioxidant attached to human red blood cells (<b>1</b>). NHS functional group attachment facilitates cell attachment via covalent amide bond formation with surface proteins (<b>2</b>). Polymer chains lacking NHS functionality were used as a control for cell surface binding (<b>3</b>). The average molecular weight of polymer chains was approximately 6.2kDa.</p