Elongated microparticles tuned for targeting hyaluronic acid delivery to specific skin strata.

OBJECTIVE: Microneedle or fractional laser applications are the most common topical delivery enhancement platforms. However, these methods of drug delivery are not skin strata specific. Drug delivery approaches which could target specific stratum of the skin remains a challenge. Elongated microparticles (EMPs) have been used in enhancing drug delivery into the skin. The aim of this study was to evaluate, for the first time, elongated silica microparticles with two different length profiles to enhance delivery of hyaluronic acid into different strata of human skin. METHODS: Two types of EMPs-long (milled EMPs) or short (etched EMPs) length ranges were characterized. A prototypical liquid formulation (Fluorescent hyaluronic acid) with and without EMP enhancement were evaluated for hyaluronic acid delivery in ex-vivo human skin. High performance liquid chromatography, Typhoon fluorescence scanning system, laser scanning confocal microscopy and reflectance confocal microscopy (RCM) were used to validate F-HA stability, visualize fluorescein in the skin, image the depth of F-HA delivery in the skin and define EMP penetration in skin strata, respectively. Statistical analysis was conducted using GraphPad Prism 6 software (GraphPad Software Inc, USA). RESULTS: Fluorescein-hyaluronic acid was stable and EMP enhanced skin penetration. RCM revealed that 'etched EMP' penetrated the skin to the stratum spinosum level. The vast majority (97.8%; p < 0.001) of the etched EMP did not penetrate completely through the viable epidermis and no obvious penetration into the dermis. In contrast, milled EMP showed 41-fold increase in penetration compared to the etched EMP but penetrated beyond the dermoepidermal junction. CONCLUSION: EMPs can enhance delivery of hyaluronic acid. Using EMPs with defined length distributions, which can be tuned for a specific stratum of the skin, can achieve targeted hyaluronic acid delivery.

OBJECTIF: Les microaiguilles ou le laser fractionné sont couramment utilisés pour augmenter l'absorption d'actif appliqué par voie topique. Toutefois, ces approches ne permettent de cibler une strate spécifique de la peau. Ainsi les méthodes permettant de cibler spécifiquement l'épiderme reste un défi. Les microparticules allongées (EMP) ont déjà été utilisé pour augmenter l'absorption cutanée. L'objectif de l'étude est d'évaluer pour la première fois, la capacité de microparticules allongées de silice (de deux longueurs différentes) à délivrer l'acide hyaluronique dans les différentes couches de la peau. MÉTHODES: Deux types d'EMP, longues (EMP broyé) ou courtes (EMP gravé), ont été caractérisées. Une formulation liquide contenant de l'acide hyaluronique marquée avec une sonde fluorescente (F-HA) a été évaluée avec et sans EMP sur peau humaine ex vivo. La chromatographie liquide haute performance, le scanner à fluorescence Typhoon, la microscopie laser confocal à balayage et la microscopie confocale par réflectance (RCM) ont été utilisées respectivement pour contrôler la stabilité de la F-HA, visualiser le signal de la fluorescéine dans la peau, imager l'absorption du F-HA dans la peau en fonction de la profondeur et caractériser la pénétration des EMP. Les analyses statistiques ont été réalisées avec le logiciel GraphPad Prims 6 (GraphPad Software Inc, USA). RÉSULTATS: L'acide hyaluronique marquée avec la fluorescéine est stable et les EMP permettent d'augmenter son absorption cutanée. Le RCM a montré que les EMP gravées pénètrent dans la peau jusqu'au niveau du stratum spinosum. La grande majorité des EMP gravés (97.8% ; p < 0,001) ne pénètre pas complétement dans l'épiderme viable et aucune pénétration mesurable dans le derme. Au contraire, les EMP broyées ont montrées une pénétration 41 fois plus importantes que les EMP gravées et peuvent aller au-delà de la jonction derme-épiderme. CONCLUSION: Les EMP peuvent augmenter l'absorption cutanée de l'acide hyaluronique. En utilisant des EMP de longueur définie et en ajustant celle-ci, il est même possible de cibler spécifiquement une strate cutanée.

Polycyclic aromatic hydrocarbons regulate the pigmentation pathway and induce DNA damage responses in keratinocytes, a process driven by systemic immunity.

BACKGROUND: Urban pollution is correlated with an increased prevalence of skin pigmentation disorders, however the physiological processes underlying this association are unclear. OBJECTIVES: To delineate the relationship between polycyclic aromatic hydrocarbons (PAHs), a key constituent of atmospheric pollution, and immunity/skin pigmentation pathways. METHODS: We exposed peripheral blood mononuclear cells (PBMC) to PAHs and performed cytokines/chemokine profiling. We then examined the effect of immune activation on pigmentation by co-culturing PBMC and Benzo(a)pyrene (BaP) with reconstructed human pigmented epidermis (RHPE). To study the mechanism, we treated keratinocytes with conditioned medium from BaP-exposed PBMC and studied DNA damage responses, aryl hydrocarbon receptor (AhR) activation and pro-pigmentation factor, proopiomelanocortin (POMC) secretion. RESULTS: PAHs induced up-regulation of inflammatory cytokines/chemokine in PBMC. Co-culturing of RHPE with PBMC+BaP resulted in increased melanin content and localization. BaP-conditioned medium significantly increased DNA damage, p53 stabilization, AhR activation and POMC secretion in keratinocytes. We found that IFNγ induced DNA damage, while TNFα and IL-8 potentiated POMC secretion in keratinocytes. Importantly, BaP-conditioned medium-induced DNA damage and POMC secretion is prevented by antioxidants vitamin E, vitamin C and sulforaphane, as well as the prototypical corticosteroid dexamethasone. Finally, vitamin C and sulforaphane enhanced the genome protective and depigmentation effects of dexamethasone, providing proof-of-concept for a combinatorial approach for the prevention and/or correction of PAH-induced pigment spots formation. CONCLUSION: Our study reveals the importance of systemic immunity in regulating PAH-induced skin pigmentation, and provide a new keratinocyte DNA damage response mechanistic target for the prevention or reversal of pollution-associated skin pigmentation.

Bone marrow-derived heparan sulfate potentiates the osteogenic activity of bone morphogenetic protein-2 (BMP-2).

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS-5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung do not generate effects as great as HS5. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively supports bone formation, and suggest its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.

The effect of human bone marrow stroma-derived heparan sulfate on the ex vivo expansion of human cord blood hematopoietic stem cells.

PURPOSE: In order to address cell dose limitations associated with the use of cord blood hematopoietic stem cell (HSC) transplantation, we explored the effect of bone marrow stroma-derived heparan sulfate (HS) on the ex vivo expansion of HSCs. METHODS: Heparan sulfate was isolated and purified from the conditioned media of human bone marrow stromal cells and used for the expansion of cord blood-derived CD34(+) cells in the presence of a cocktail of cytokines. RESULTS: The number of myeloid lineage-committed progenitor cells was increased at low dosage of HS as illustrated by an increase in the total number of colony-forming cells (CFC) and colonies of erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) precursors. Notably, the stroma-derived HS did not alter the growth of CD34(+) HSCs or negatively affect the levels of various HSC phenotypic markers after expansion. CONCLUSIONS: This study shows that HS secreted into solution by stromal cells has the capacity to support hematopoietic cytokines in the maintenance and expansion of HSCs. The incorporation of stroma-derived HS as a reagent may improve the efficacy of cord blood HSC transplantation by enhancing the number of committed cells and accelerating the rate of engraftment.

Sequential biochemical and mechanical stimulation in the development of tissue-engineered ligaments.

Application of stimuli in sequence to developing cultures in vitro offers the potential to intricately direct cell development and differentiation by following the template of native tissue behavior. We hypothesize that administration of mechanical stimulation at the peak of growth factor-induced cell activity will differentiate bone marrow stromal cells (BMSCs) along a fibroblast lineage and enhance in vitro ligament development through enhanced matrix ingrowth, matrix metalloproteinase-2 (MMP-2) production, collagen type I production, and extracellular matrix (ECM) alignment. BMSC-seeded silk matrices were cultured in a static growth-factor-free environment for 5 days prior to loading into bioreactor vessels to first establish an appropriate dynamic rotational regime, as determined through assessment of cell activity, histology, and surface topography. Once the regime was determined, seeded matrices initially cultured in basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or growth-factor-free control medium for 5 days were loaded into the bioreactor for 9 days of mechanical stimulation. Our findings indicated that the sequential application of mechanical stimulation following growth factor supplemented static culture-induced cell differentiation toward a fibroblast lineage, enhancing matrix ingrowth, cell and ECM alignment, and total collagen type I produced compared to respective static cultures. The current results suggest a dynamic culturing regime in the development of engineered tissues.

Monitoring mesenchymal stromal cell developmental stage to apply on-time mechanical stimulation for ligament tissue engineering.

To evaluate the appropriate time frame for applying mechanical stimuli to induce mesenchymal stromal cell (MSC) differentiation for ligament tissue engineering, developmental cell phenotypes were monitored during a period of in vitro culture. MSCs were seeded onto surface-modified silk fibroin fiber matrices and cultured in Petri dishes for 15 days. Cell metabolic activity, morphology, and gene expression of extracellular matrix (ECM) proteins (collagen type I and III and fibronectin), ECM receptors (integrins alpha-2, alpha-5, and beta-1), and heat-shock protein 70 (HSP-70) were monitored during the culture of MSC. MSCs showed fluctuations in cell metabolic activity, ECM, integrin, and HSP-70 transcription potentially correlating to innate developmental processes. Cellular response to mechanical stimulation was dependent on the stage of cell development. At day 9, when levels of cell metabolic activity, ECM, integrin, and HSP-70 transcription peaked, mechanical stimulation increased MSC metabolic activity, alignment, and collagen production. Mechanical stimulation applied at day 1 and 3 showed detrimental effects on MSCs seeded on silk matrices. The results presented in this study identify a unique correlation between innate MSC development processes on a surface-modified silk matrix and dynamic environmental signaling.

Characterization of transcript levels for matrix molecules and proteases in ruptured human anterior cruciate ligaments.

An improved understanding of cellular responses during normal anterior cruciate ligament (ACL) function or repair is essential for clinical assessments, understanding ligament biology, and the implementation of tissue engineering strategies. The present study utilized quantitative real-time RT-PCR combined with univariate and multivariate statistical analyses to establish a quantitative database of marker transcript expression that can provide a "blueprint" of ACL wound healing. Selected markers (collagen types I and III, biglycan, decorin, MMP-1, MMP-2, MMP-9, and TIMP-1) were assessed from 33 torn ACLs harvested during reconstructive surgery. Trends were observed between postinjury period and marker expressions. Significant correlations between marker expression existed and were most prominent between collagen types I and III. Canonical correlation analysis established a relationship between patient demographics and a combination of all marker expressions. The currently observed trends and correlations may assist in identifying appropriate tissue samples and provide a baseline information of marker expression level that can support in vitro optimization of environmental cues for ligament tissue engineering application.

Growth factor induced fibroblast differentiation from human bone marrow stromal cells in vitro.

Utilizing a two-dimensional tissue culture plastic screening system and a fractional factorial design, specific media formulations and growth factor combinations were determined that support human bone marrow stromal cell (BMSC) differentiation toward fibroblast characteristics for utilization in tissue engineering, specifically cell morphology and alignment, metabolic activity, abundant expression of collagen types I and III, and negligible expression of other tissue-specific markers. BMSCs were cultured for up to 14 days on tissue culture plastic, supplemented with Dulbecco's Minimal Essential Medium (DMEM)/10% FBS or Advanced DMEM(ADMEM)/5% FBS. Each medium base was supplemented with one of nine possible growth factor combinations and ascorbate-2-phosphate (Asc-2-P) for the duration of culture. ADMEM supported comparable cell viability with half the serum content of the DMEM formulation. Asc-2-P was potent in promoting BMSC proliferation, in the absence of a mitogen, supporting significant increases in cell activity over 14 days of culture. DMEM promoted significant increases in cell viability for 7 of 9 growth factor groups when compared to their ADMEM counterparts. ADMEM, however, promoted increased cell transcript and protein expression, as 5 of 9 growth factor combinations induced a 200% increase in collagen type I versus equivalent DMEM cultures. Cell morphology and collagen type I immunostaining, when assessed in context of MTT and RNA results, identified 3 growth factor and medium combinations that supported fibroblast differentiation for future development of ligament tissue in vitro.

Matrix metalloproteinases and their clinical applications in orthopaedics.

Imbalance in the expression of matrix metalloproteinases and their inhibitors contribute considerably to abnormal connective tissue degradation prevalent in various orthopaedic joint diseases such as rheumatoid arthritis and osteoarthritis. Matrix metalloproteinase expression has been detected in ligament, tendon, and cartilage tissues in the joint. They are known to contribute to the development, remodeling, and maintenance of healthy tissue through their ability to cleave a wide range of extracellular matrix substrates. Their role has been extended to cell growth, migration, differentiation, and apoptosis. In orthopaedics, their clinical applications constantly are being explored. The multiple steps in matrix metalloproteinase regulation offer potential targets for inhibition, useful in drug therapy. The correlation between matrix metalloproteinases and progression in joint erosion presents potential prognostic and diagnostic tools in rheumatoid arthritis. Matrix metalloproteinases also can be incorporated into scaffold design to control the degradation rate of engineered tissue constructs. This current review aims to summarize and emphasize the importance of matrix metalloproteinases and their natural inhibitors in the maturation of musculoskeletal tissue through matrix remodeling and, therefore, in the generation of a new clinical potential in orthopaedics.