Therapeutic delivery of CCL2 modulates immune response and restores host-microbe homeostasis.

Many chronic inflammatory diseases are attributed to disturbances in host-microbe interactions, which drive immune-mediated tissue damage. Depending on the anatomic setting, a chronic inflammatory disease can exert unique local and systemic influences, which provide an exceptional opportunity for understanding disease mechanism and testing therapeutic interventions. The oral cavity is an easily accessible environment that allows for protective interventions aiming at modulating the immune response to control disease processes driven by a breakdown of host-microbe homeostasis. Periodontal disease (PD) is a prevalent condition in which quantitative and qualitative changes of the oral microbiota (dysbiosis) trigger nonresolving chronic inflammation, progressive bone loss, and ultimately tooth loss. Here, we demonstrate the therapeutic benefit of local sustained delivery of the myeloid-recruiting chemokine (C-C motif) ligand 2 (CCL2) in murine ligature-induced PD using clinically relevant models as a preventive, interventional, or reparative therapy. Local delivery of CCL2 into the periodontium inhibited bone loss and accelerated bone gain that could be ascribed to reduced osteoclasts numbers. CCL2 treatment up-regulated M2-macrophage and downregulated proinflammatory and pro-osteoclastic markers. Furthermore, single-cell ribonucleic acid (RNA) sequencing indicated that CCL2 therapy reversed disease-associated transcriptomic profiles of murine gingival macrophages via inhibiting the triggering receptor expressed on myeloid cells-1 (TREM-1) signaling in classically activated macrophages and inducing protein kinase A (PKA) signaling in infiltrating macrophages. Finally, 16S ribosomal ribonucleic acid (rRNA) sequencing showed mitigation of microbial dysbiosis in the periodontium that correlated with a reduction in microbial load in CCL2-treated mice. This study reveals a novel protective effect of CCL2 local delivery in PD as a model for chronic inflammatory diseases caused by a disturbance in host-microbe homeostasis.

Local Sustained Delivery of Anti-IL-17A Antibodies Limits Inflammatory Bone Loss in Murine Experimental Periodontitis.

Periodontal disease (PD) is a chronic destructive inflammatory disease of the tooth-supporting structures that leads to tooth loss at its advanced stages. Although the disease is initiated by a complex organization of oral microorganisms in the form of a plaque biofilm, it is the uncontrolled immune response to periodontal pathogens that fuels periodontal tissue destruction. IL-17A has been identified as a key cytokine in the pathogenesis of PD. Despite its well documented role in host defense against invading pathogens at oral barrier sites, IL-17A-mediated signaling can also lead to a detrimental inflammatory response, causing periodontal bone destruction. In this study, we developed a local sustained delivery system that restrains IL-17A hyperactivity in periodontal tissues by incorporating neutralizing anti-IL-17A Abs in poly(lactic-coglycolic) acid microparticles (MP). This formulation allowed for controlled release of anti-IL-17A in the periodontium of mice with ligature-induced PD. Local delivery of anti-IL-17A MP after murine PD induction inhibited alveolar bone loss and osteoclastic activity. The anti-IL-17A MP formulation also decreased expression of IL-6, an IL-17A target gene known to induce bone resorption in periodontal tissues. This study demonstrates proof of concept that local and sustained release of IL-17A Abs constitutes a promising therapeutic strategy for PD and may be applicable to other osteolytic bone diseases mediated by IL-17A-driven inflammation.

Sterilization and Biologic Monitoring in Private Dental Clinics in Lebanon.

AIM: The aim of the present study was to evaluate sterilization practices and effectiveness in the Lebanese private dental sector and identify potential factors contributing to sterilization failure. MATERIALS AND METHODS: A 13-item questionnaire consisting of four demographic/professional questions and nine questions related to sterilization practices along with self-contained biologic indicators (SCBIs) were delivered to a representative sample of Lebanese private offices. Univariate statistics and bivariate analyses were performed to compare sterilization failure rates according to demographic, professional, and sterilization-related conditions. RESULTS: Out of the 560 dentists contacted, 205 dentists returned the completed questionnaires and undamaged processed SCBIs. The tested autoclaves (n = 134) were mostly dynamic air removal (69.4%) and had a mean age of 10.5 ± 6.9 years. The dry heat ovens (n = 71) were all static air and had 12.9 ± 8.1 years. The dental assistants performed the routine sterilization procedures in nearly 62% of the practices and sterilization cycles were run 4 to 6 times per week in 75% of the offices. Correct temperature/time ratios were applied in 97% of the autoclaves and 80.3% of the ovens. Few dental practices reported having preventive maintenance (17.9% for the autoclaves and 14.1% for the ovens). Routine monitoring of sterilizer efficacy was infrequently performed and was mostly conducted using physical indicators. Sterilization failure rate was higher for the ovens (16.9%) than for the autoclaves (7.5%). Incorrect temperature/time ratio was the main significant factor associated with sterilization failures. CONCLUSION: The present study demonstrated a relatively high rate of sterilization failures in the Lebanese private dental sector and identified the human error in setting sterilization cycle parameters as the predominant cause of failure. These findings should prompt actions toward increasing knowledge of the sterilization processes and their monitoring among dental professionals and improving the quality control of sterilization through collaborative efforts among health authorities, dental schools, and associations. CLINICAL SIGNIFICANCE: This study presents the first published data relative to sterilization practices and effectiveness in private Lebanese dental offices and provides a rationale to implement biologic monitoring protocols in Lebanon as long practiced in developed countries.

Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes.

The hallmark of periodontal disease is the progressive destruction of gingival soft tissue and alveolar bone, which is initiated by inflammation in response to an invasive and persistent bacterial insult. In recent years, it has become apparent that this tissue destruction is associated with a decrease in local regulatory processes, including a decrease of forkhead box P3-expressing regulatory lymphocytes. Accordingly, we developed a controlled release system capable of generating a steady release of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22), composed of a degradable polymer with a proven track record of clinical translation, poly(lactic-co-glycolic) acid. We have previously shown that this sustained presentation of CCL22 from a point source effectively recruits regulatory T cells (Tregs) to the site of injection. Following administration of the Treg-recruiting formulation to the gingivae in murine experimental periodontitis, we observed increases in hallmark Treg-associated anti-inflammatory molecules, a decrease of proinflammatory cytokines, and a marked reduction in alveolar bone resorption. Furthermore, application of the Treg-recruiting formulation (fabricated with human CCL22) in ligature-induced periodontitis in beagle dogs leads to reduced clinical measures of inflammation and less alveolar bone loss under severe inflammatory conditions in the presence of a diverse periodontopathogen milieu.

Regeneration of periosteum by human bone marrow stromal cell sheets.

PURPOSE: The presence of a functional periosteum accelerates healing in bone defects by providing a source of progenitor cells that aid in repair. We hypothesized that bone marrow stromal cell (BMSC) sheets could be used to engineer functional periosteal tissues. MATERIALS AND METHODS: BMSCs were cultured to hyperconfluence and produced sufficient extracellular matrix to form robust tissue sheets. The sheets were wrapped around calcium phosphate pellets and implanted subcutaneously in mice for 8 weeks. Histologic comparisons were made between calcium phosphate samples with and without BMSC sheet wraps. Bone and periosteum formation were analyzed through tissue morphology and tissue-specific protein expression. RESULTS: Calcium phosphate pellets wrapped in BMSC sheets regenerated a bone-like tissue, but pellets lacking the cell sheet wrap did not. The bone-like tissue seen on the calcium phosphate scaffolds wrapped with the BMSC sheets was enclosed within a periosteum-like tissue characterized morphologically and through expression of periostin. CONCLUSIONS: These data indicate that cell sheet technology has potential for regenerating a functional periosteum-like tissue that could aid in future orthopedic therapy.

Expression of phosphophoryn is sufficient for the induction of matrix mineralization by mammalian cells.

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.

Dentin matrix protein 1 (DMP1) signals via cell surface integrin.

Dentin matrix phosphoprotein 1 (DMP1) is a non-collagenous, acidic extracellular matrix protein expressed chiefly in bone and dentin. We examined the DMP1 ability to engage cell-surface receptors and subsequently activate intracellular signaling pathways. Our data indeed show that the presence of extracellular DMP1 triggers focal adhesion point formation in human mesenchymal stem cells and osteoblast-like cells. We determine that DMP1 acts via interaction with αvß3 integrin and stimulates phosphorylation of focal adhesion kinase. Further biochemical characterization confirms the activation of downstream effectors of the MAPK pathways, namely ERK and JNK, after DMP1 treatment. This activation is specifically inhibitable and can also be blocked by the addition of anti-αvß3 integrin antibody. Furthermore, we show that extracellular treatment with DMP1 stimulates the translocation of phosphorylated JNK to the nucleus and a concomitant up-regulation of transcriptional activation by phosphorylated c-Jun. The evidence presented here indicates that DMP1 is specifically involved in signaling via extracellular matrix-cell surface interaction. Combined with the published DMP1-null data (Feng, J. Q., Ward, L. M., Liu, S., Lu, Y., Xie, Y., Yuan, B., Yu, X., Rauch, F., Davis, S. I., Zhang, S., Rios, H., Drezner, M. K., Quarles, L. D., Bonewald, L. F., and White, K. E. (2006) Nat. Genet. 38, 1310-1315) it can be hypothesized that DMP1 could be a key effector of ECM-osteocyte signaling.

Droplet-based microsystems as novel assessment tools for oral microbial dynamics.

The human microbiome comprises thousands of microbial species that live in and on the body and play critical roles in human health and disease. Recent findings on the interplay among members of the oral microbiome, defined by a personalized set of microorganisms, have elucidated the role of bacteria and yeasts in oral health and diseases including dental caries, halitosis, and periodontal infections. However, the majority of these studies rely on traditional culturing methods which are limited in their ability of replicating the oral microenvironment, and therefore fail to evaluate key microbial interactions in microbiome dynamics. Novel culturing methods have emerged to address this shortcoming. Here, we reviewed the potential of droplet-based microfluidics as an alternative approach for culturing microorganisms and assessing the oral microbiome dynamics. We discussed the state of the art and recent progress in the field of oral microbiology. Although at its infancy, droplet-based microtechnology presents an interesting potential for elucidating oral microbial dynamics and pathophysiology. We highlight how new findings provided by current microfluidic-based methodologies could advance the investigation of the oral microbiome. We anticipate that our work involving the droplet-based microfluidic technique with a semipermeable membrane will lay the foundations for future microbial dynamics studies and further expand the knowledge of the oral microbiome and its implication in oral health.

Local induction of regulatory T cells prevents inflammatory bone loss in ligature-induced experimental periodontitis in mice.

Periodontitis (periodontal disease) is a highly prevalent disease, affecting over 65 million adults in the United States alone. Characterized by an overburden of invasive bacteria, gum inflammation and plaque buildup, over time, these symptoms can result in severe loss of gingival tissue attachment, bone resorption and even tooth loss. Although current treatments (local antibiotics and scaling and root planing procedures) target the bacterial dysbiosis, they do not address the underlying inflammatory imbalance in the periodontium. In the healthy steady state, the body naturally combats destructive, imbalanced inflammatory responses through regulatory pathways mediated by cells such as regulatory T cells (Tregs). Consequently, we hypothesized that local enrichment of regulatory lymphocytes (Tregs) could restore local, immunological homeostasis and prevent the main outcome of bone loss. Accordingly, we locally delivered a combination of TGFß, Rapamycin, and IL2 microspheres in a ligature-induced murine periodontitis model. Herein, we have demonstrated this preventative treatment decreases alveolar bone loss, increases the local ratio of Tregs to T effector cells and changes the local microenvironment's expression of inflammatory and regenerative markers. Ultimately, these Treg-inducing microspheres appear promising as a method to improve periodontitis outcomes and may be able to serve as a platform delivery system to treat other inflammatory diseases.

Possible role of DMP1 in dentin mineralization.

Dentin Matrix Protein 1 (DMP1), the essential noncollagenous proteins in dentin and bone, is believed to play an important role in the mineralization of these tissues, although the mechanisms of its action are not fully understood. To gain insight into DMP1 functions in dentin mineralization we have performed immunomapping of DMP1 in fully mineralized rat incisors and in vitro calcium phosphate mineralization experiments in the presence of DMP1. DMP1 immunofluorescene was localized in peritubular dentin (PTD) and along the dentin-enamel boundary. In vitro phosphorylated DMP1 induced the formation of parallel arrays of crystallites with their c-axes co-aligned. Such crystalline arrangement is a hallmark of mineralized collagen fibrils of bone and dentin. Interestingly, in DMP1-rich PTD, which lacks collagen fibrils, the crystals are organized in a similar manner. Based on our findings we hypothesize, that in vivo DMP1 controls the mineral organization outside of the collagen fibrils and plays a major role in the mineralization of PTD.

Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization.

The SIBLING (small integrin-binding ligand N-linked glycoproteins) family is the major group of noncollagenous proteins in bone and dentin. These extremely acidic and highly phosphorylated extracellular proteins play critical roles in the formation of collagenous mineralized tissues. Whereas the lack of individual SIBLINGs causes significant mineralization defects in vivo, none of them led to a complete cessation of mineralization suggesting that these proteins have overlapping functions. To assess whether different SIBLINGs regulate biomineralization in a similar manner and how phosphorylation impacts their activity, we studied the effects of two SIBLINGs, dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP), on mineral morphology and organization in vitro. Our results demonstrate distinct differences in the effects of these proteins on mineralization. We show that phosphorylation has a profound effect on the regulation of mineralization by both proteins. Specifically, both phosphorylated proteins facilitated organized mineralization of collagen fibrils and phosphorylated DMP1-induced formation of organized mineral bundles in the absence of collagen. In summary, these results indicate that the primary structure and phosphorylation uniquely determine functions of individual SIBLINGs in regulation of mineral morphology and organization.

Biorelevant and screening dissolution methods for minocycline hydrochloride microspheres intended for periodontal administration.

Currently, there is no compendial-level method to assess dissolution of particulate systems administered in the periodontal pocket. This work seeks to develop dissolution methods for extended release poly(lactic-co-glycolic acid) (PLGA) microspheres applied in the periodontal pocket. Arestin®, PLGA microspheres containing minocycline hydrochloride (MIN), is indicated for reduction of pocket depth in adult periodontitis. Utilizing Arestin® as a model product, two dissolution methods were developed: a dialysis set-up using USP apparatus 4 and a novel apparatus fabricated to simulate in vivo environment of the periodontal pocket. In the biorelevant method, the microspheres were dispersed in 250 µL of simulated gingival crevicular fluid (sGCF) which was enclosed in a custom-made dialysis enclosure. sGCF was continuously delivered to the device at a biorelevant flow rate and was collected daily for drug content analysis using UPLC. Both methods could discriminate release characteristics of a panel of MIN-loaded PLGA microspheres that differed in composition and process conditions. A mechanistic model was developed, which satisfactorily explained the release profiles observed using both dissolution methods. The developed methods may have the potential to be used as routine quality control tools to ensure batch-to-batch consistency and to support evaluation of bioequivalence for periodontal microspheres.

Effect of the Periapical "Inflammatory Plug" on Dental Pulp Regeneration: A Histologic In Vivo Study.

INTRODUCTION: In the current study, we investigate the effect of the inflammation occupying the apical foramen-a phenomenon we refer to as "inflammatory plug"-on the regenerative potential of a root canal therapy. METHODS: We performed root canal treatment (RCT) in 12 canine root canals while aseptically instrumenting the apex to a 0.5-mm-wide foramen and obturating the canals with the following materials: collagen sponge, platelet-rich fibrin, and blood clot (no material introduced). RESULTS: We were successful in maintaining the integrity of the periapical tissue in 8 of 12 RCTs. Injury to the periapical tissue occurred during the remaining 4 RCTs, which initiated inflammation accompanied by bone and dentin resorption. Our histologic analyses showed that the resulting inflammatory plug contained abundant M1 macrophages and was associated with an absence of intracanal cellular infiltration. On the contrary, noninflamed samples showed signs of repair, as indicated by the migration of periapical cells throughout the root canal. CONCLUSIONS: We conclude that controlling periapical inflammation is key while attempting to achieve dental pulp regeneration.

Vasoactive Intestinal Peptide Immunoregulatory Role at the Periapex: Associative and Mechanistic Evidences from Human and Experimental Periapical Lesions.

INTRODUCTION: The balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions. METHODS: Periapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects. RESULTS: VIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4. CONCLUSIONS: Our results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells.

In vivo quantification of hydrogen gas concentration in bone marrow surrounding magnesium fracture fixation hardware using an electrochemical hydrogen gas sensor.

Magnesium (Mg) medical devices are currently being marketed for orthopedic applications and have a complex degradation process which includes the evolution of hydrogen gas (H2). The effect of H2 exposure on relevant cell types has not been studied; and the concentration surrounding degrading Mg devices has not been quantified to enable such mechanistic studies. A simple and effective method to measure the concentration of H2 in varying microenvironments surrounding Mg implants is the first step to understanding the biological impact of H2 on these cells. Here, the in vivo measurement of H2 surrounding fracture fixation devices implanted in vivo is demonstrated. An electrochemical H2 microsensor detected increased levels of H2 at three anatomical sites with a response time of about 30 s. The sensor showed the H2 concentration in the bone marrow at 1 week post-implantation (1460 ±â€¯320 µM) to be much higher than measured in the subcutaneous tissue (550 ±â€¯210 µM) and at the skin surface (120 ±â€¯50 µM). Additionally, the H2 concentrations measured in the bone marrow exceeded the concentration in a H2 saturated water solution (∼800 µM). These results suggest that H2 emanating from Mg implants in bone during degradation pass through the bone marrow and become at least partially trapped because of slow permeation through the bone. This study is the first to identify H2 concentrations in the bone marrow environment and will enable in vitro experiments to be executed at clinically relevant H2 concentrations to explore possible biological effects of H2 exposure. STATEMENT OF SIGNIFICANCE: An electrochemical H2 sensor was used to monitor the degradation of a Mg fracture fixation system in a lapine ulna fracture model. Interestingly, the H2 concentration in the bone marrow is 82% higher than H2 saturated water solution. This suggests H2 generated in situ is trapped in the bone marrow and bone is less permeable than the surrounding tissues. The detectable H2 at the rabbit skin also demonstrates a H2 sensor's ability to monitor the degradation process under thin layers of tissue. H2 sensing shows promise as a tool for monitoring the degradation of Mg alloy in vivo and creating in vitro test beds to more mechanistically evaluate the effects of varying H2 concentrations on cell types relevant to osteogenesis.

Infection Control Measures in Private Dental Clinics in Lebanon.

PURPOSE: Evaluate infection control knowledge, attitude, and practice in Lebanese private dental clinics. MATERIALS AND METHODS: A survey including 46 questions related to routine safety procedures was sent to 1150 Lebanese dentists between July 1st and 2nd, 2015. The study sample was selected from the database of registered dentists based on a proportional random sampling ensuring equitable representation of the 5 geographic regions of Lebanon. A subset of 29 questions was used to generate an overall score of compliance (excellent, good, fair, and poor). Comparisons according to gender, type, region, and years of practice were performed. RESULTS: 417 dentists returned the completed questionnaires. 96% expressed concern about infection transmission, 90.6% were vaccinated against Hepatitis B, and 61.8% asked routinely about patients medical history. Only 43% used protective eyewear. Although most dentists (65%) used autoclaves, dry heat was still used. Significant correlations were found between gender and use of personal protective equipment. Less compliance was shown by clinicians with fewer years of experience. In the overall compliance questionnaire, the mean percentage of correct answers was roughly 54% with <5% of the practitioners scoring "excellent." Conclusions. The study found inadequacy of compliance in private Lebanese dental clinics necessitating improved educational training and sustained monitoring by regulatory bodies.

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool.

INTRODUCTION: The pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions. METHODS: Periapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti-glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array). RESULTS: Treg inhibition by anti-glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression. CONCLUSIONS: Because the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

Nanostructured calcium phosphates for biomedical applications: novel synthesis and characterization.

Materials play a key role in several biomedical applications, and it is imperative that both the materials and biological aspects are clearly understood for attaining a successful biological outcome. This paper illustrates our approach to implement calcium phosphates as gene delivery agents. Calcium phosphates (CaP) belong to the family of biocompatible apatites and there are several CaP phases, the most ubiquitous being hydroxyapatite (HAp, Ca(10)(PO(4))(6)(OH)(2). Other CaP structures include brushite (B, CaHPO(4).2H(2)O) and tricalcium phosphate (TCP, Ca(3)(PO(4))(2)). Several low and high temperature approaches have been reported for synthesizing HAp and brushite, while TCP is primarily synthesized using high temperature methods. Novel low temperature chemical methods have been developed by us to synthesize nanostructured HAp, brushite and TCP phases. The new low temperature approach results in the formation of stoichiometric and nanosized HAp under physiological conditions. Moreover, the synthesis methods were designed to be biocompatible with biological systems such as cells, DNA and proteins so that the CaP structures can be studied for gene delivery. The use of HAp type CaP phases for gene delivery is well known but to our knowledge, other forms of CaP have not been studied for gene delivery due to the lack of a biocompatible synthesis method. In addition to the biocompatible synthesis of CaP structures, we have also performed ion substitution that would provide us the appropriate tools to study the DNA-to-particle interactions and assess how these ionic substitutions would affect the level of DNA uptake by the cell and then its release to the cell nucleus. Substitution of calcium by 14% magnesium results in the formation of crystalline ( approximately 20 mum) brushite platelets that remains stable at pH 7.5. Further substitution results in unique nanostructured spherical morphologies of brushite from which rosette shaped high specific surface area ( approximately 200 m(2)/g) nanocrystals ( approximately 80 nm) of beta-TCMP phase can be grown. The novelty lies in the formation of stable phases of HAp, brushite and beta-TCMP under physiological conditions making them potential candidates for use as carriers for non-viral gene delivery or more generally in biological systems. The resultant nanocrystalline phosphates have been characterized for their structure, morphology, thermal stability, and composition. Results of the in vitro transfection are also described.

Porous magnesium/PLGA composite scaffolds for enhanced bone regeneration following tooth extraction.

Sixty percent of implant-supported dental prostheses require bone grafting to enhance bone quantity and quality prior to implant placement. We have developed a metallic magnesium particle/PLGA composite scaffold to overcome the limitations of currently used dental bone grafting materials. This is the first report of porous metallic magnesium/PLGA scaffolds synthesized using a solvent casting, salt leaching method. We found that incorporation of varying amounts of magnesium into the PLGA scaffolds increased the compressive strength and modulus, as well as provided a porous structure suitable for cell infiltration, as measured by mercury intrusion porosimetry. Additionally, combining basic-degrading magnesium with acidic-degrading PLGA led to an overall pH buffering effect and long-term release of magnesium over the course of a 10-week degradation assay, as measured with inductively coupled plasma-atomic emission spectroscopy. Using an indirect proliferation assay adapted from ISO 10993:5, it was found that extracts of medium from degrading magnesium/PLGA scaffolds increased bone marrow stromal cell proliferation in vitro, a phenomenon observed by other groups investigating magnesium's impact on cells. Finally, magnesium/PLGA scaffold biocompatibility was assessed in a canine socket preservation model. Micro-computed tomography and histological analysis showed the magnesium/PLGA scaffolds to be safer and more effective at preserving bone height than empty controls. Three-dimensional magnesium/PLGA composite scaffolds show promise for dental socket preservation and also, potentially, orthopedic bone regeneration. These scaffolds could decrease inflammation observed with clinically used PLGA devices, as well as enhance osteogenesis, as observed with previously studied magnesium devices.