Impact of interstitial flow on cartilage matrix synthesis and NF-kB transcription factor mRNA expression in a novel perfusion bioreactor.

This work is focused on designing an easy-to-use novel perfusion system for articular cartilage (AC) tissue engineering and using it to elucidate the mechanism by which interstitial shear upregulates matrix synthesis by articular chondrocytes (AChs). Porous chitosan-agarose (CHAG) scaffolds were synthesized and compared to bulk agarose (AG) scaffolds. Both scaffolds were seeded with osteoarthritic human AChs and cultured in a novel perfusion system with a medium flow velocity of 0.33 mm/s corresponding to 0.4 mPa surfice shear and 40 mPa CHAG interstitial shear. While there were no statistical differences in cell viability for perfusion versus static cultures for either scaffold type, CHAG scaffolds exhibited a 3.3-fold higher (p < 0.005) cell viability compared to AG scaffold cultures. Effects of combined superficial and interstitial perfusion for CHAG showed 150- and 45-fold (p < 0.0001) increases in total collagen (COL) and 13- and 2.2-fold (p < 0.001) increases in glycosaminoglycans (GAGs) over AG non-perfusion and perfusion cultures, respectively, and a 1.5-fold and 3.6-fold (p < 0.005) increase over non-perfusion CHAG cultures. Contrasting CHAG perfusion and static cultures, chondrogenic gene comparisons showed a 3.5-fold increase in collagen type II/type I (COL2A1/COL1A1) mRNA ratio (p < 0.05), and a 1.3-fold increase in aggrecan mRNA. Observed effects are linked to NF-κB signal transduction pathway inhibition as confirmed by a 3.2-fold (p < 0.05) reduction of NF-κB mRNA expression upon exposure to perfusion. Our results demonstrate that pores play a critical role in improving cell viability and that interstitial flow caused by medium perfusion through the porous scaffolds enhances the expression of chondrogenic genes and extracellular matrix through downregulating NF-κB1.

Three-Dimensional Biomaterials with Spatiotemporal Control for Regenerative Tissue Engineering.

At Morehouse College, one of the nation's top liberal arts historically black colleges and universities (HBCU) for African American men, research experiences are used to enhance the liberal arts educational experience. Securing research funding to train HBCU students is highly competitive and challenging due to the review process that is typically vetted by scientists from research-intensive universities who may not be familiar with the HBCU enterprise that may be comprised of insolvent infrastructures. In this Account, the synthesis and preparation of synthetic polymeric biomaterials that are used to facilitate or support changes in biological processes, enhance mechanical properties, and foster tissue growth in three dimensions (3D) under disease conditions will be discussed. The use of biomaterials to help control biological processes in disease states is limited. Hence, the fabrication of 3D scaffolds with chemical variability to grow or repair damaged tissues by inhibiting molecular pathways shows promise by controlling the cellular response to recapitulate 3D tissues and organs. The Mendenhall laboratory at Morehouse College uses 3D biomaterials to solve biological problems by probing cellular mechanistic pathways using natural products and nanoparticles. Toward this end, we have fabricated and manufactured 3D biomaterial scaffolds using chemical strategies to mitigate biological processes to help restore pristine tissue properties. Hydrogels are 3D polymeric matrixes that swell in aqueous environments and support cell growth that later infuriates the 3D matrix to create new tissue(s). In contrast, electrospun fibers use high electric fields to create porous 3D polymeric structures that can be used to create 3D tissue molds.The synergistic use of 3D biomaterial templates that can inhibit cellular damage while providing a mechanically strong scaffold to support regenerative tissue growth is essential to creating the next generation of biomaterials. This approach will require foresight using tools from synthetic biology, molecular biology, autonomous processes, advanced biomanufacturing, and machine learning (ML). The use of several biomaterials has been explored by the Mendenhall laboratory to design, prepare, fabricate, characterize, and evaluate 3D electrospun fibers and hydrogels containing hybrid compositions of polylactic acid (PLA), poly(n-vinylcaprolactam) (PVCL), cellulose acetate (CA), and methacrylated hyaluronic acid (meHA). This work contributed to the newly fabricated PVCL-CA fibers with morphological changes and nanoscale fiber hydrophobic surface properties. While the use of electrospun fibers can create hierarchal scaffolds for bone tissue engineering, the use of injectable gels for nonporous tissues such as articular cartilage presents another compelling biomaterial challenge. Using graft polymerization, we prepared PVLC-graft-HA and studied the effect of lower critical solution temperatures (LCSTs), gelation temperatures, and mechanical properties using temperature-controlled rheology. Additionally, we reported that articular cartilage (chondrocyte) cells seeded in PVCL-g-HA gels and incubated at hypoxia 1% O2 produced a 10-fold increase in extracellular matrix proteins (collagen) after 10 days. This work supported exploring new approaches to protecting chondrocyte cells under hypoxia using a 3D scaffold technology.

Interleukin 1ß and lipopolysaccharides induction dictate chondrocyte morphological properties and reduce cellular roughness and adhesion energy comparatively.

Osteoarthritis (OA) is a whole joint disease marked by the degradation of the articular cartilage (AC) tissue, chronic inflammation, and bone remodeling. Upon AC's injury, proinflammatory mediators including interleukin 1ß (IL1ß) and lipopolysaccharides (LPS) play major roles in the onset and progression of OA. The objective of this study was to mechanistically detect and compare the effects of IL1ß and LPS, separately, on the morphological and nanomechanical properties of bovine chondrocytes. Cells were seeded overnight in a full serum medium and the next day divided into three main groups: A negative control (NC) of a reduced serum medium and 10 ng/ml IL1ß or 10 ng/ml LPS-modified media. Cells were induced for 24 h. Nanomechanical properties (elastic modulus and adhesion energy) and roughness were quantified using atomic force microscopy. Nitric oxide, prostaglandin 2 (PGE2), and matrix metalloproteinases 3 (MMP3) contents; viability of cells; and extracellular matrix components were quantified. Our data revealed that viability of the cells was not affected by inflammatory induction and IL1ß induction increased PGE2. Elastic moduli of cells were similar among IL1ß and NC while LPS significantly decreased the elasticity compared to NC. IL1ß induction resulted in least cellular roughness while LPS induction resulted in least adhesion energy compared to NC. Our images suggest that IL1ß and LPS inflammation affect cellular morphology with cytoskeleton rearrangements and the presence of stress fibers. Finally, our results suggest that the two investigated inflammatory mediators modulated chondrocytes' immediate responses to inflammation in variable ways.

From Chondrocytes to Chondrons, Maintenance of Phenotype and Matrix Production in a Composite 3D Hydrogel Scaffold.

Osteoarthritis (OA) is a degenerative disease characterized by articular cartilage (AC) degradation that affects more than 30 million people in the USA. OA is managed with symptom-alleviating medications. Matrix-assisted autologous chondrocyte transplantation (MACT) is a tissue-engineered option, but current products are expensive and lack mechanical tunability or processability to match defect mechanical properties and anatomical shapes. Here, we explore the efficacy of a biocompatible hydrogel-based scaffold composed of sodium alginate, gelatin, and gum Arabic-referred to by SA-GEL-GA-to support bovine articular chondrocyte (bAChs) proliferation, pericellular matrix (PCM), and extracellular matrix (ECM) production. bAChs were grown for 45 days in SA-GEL-GA. Their viability, their live/dead status, histological staining, biochemical assays for glycosaminoglycans (GAGs) and collagen, atomic force microscopy (AFM) imaging, and immunofluorescence staining of collagen I, collagen II, aggrecan, and CD44 were assessed. We found that SA-GEL-GA was not cytotoxic, induced cellular proliferation by 6.1-fold while maintaining a round morphology, and supported ECM deposition by producing 3.9-fold more GAG compared to day 0. bAChs transformed into chondrons and produced a PCM enriched with collagen II (3.4-fold), aggrecan (1.7-fold), and CD44 (1.3-fold) compared to day 0. In summary, SA-GEL-GA supported the proliferation, ECM production, and PCM production of bAChs in vitro.

Sex-Specific Reduction in Inflammation of Osteoarthritic Human Chondrocytes and Nutraceutical-Dependent Extracellular Matrix Formation.

INTRODUCTION: The aim of this study was to investigate the ability of osteoarthritic human chondrocytes to produce articular cartilage (AC) tissues with a reduced inflammatory environment in response to 4 anti-inflammatory nutraceuticals: alpha-tocopherol (Alpha), gallic acid (G), ascorbic acid (AA), and catechin hydrate (C). METHODS: Chondrocytes isolated from patients who underwent total knee arthroplasty surgeries were divided into groups (9 male; mean age, 66.2 ± 3.5 years and 11 female; mean age, 64.2 ± 3.1 years). Cells were cultured based on sex and supplemented with either a negative control (NC) medium or NC plus one of the nutraceuticals at a concentration of 50 µM. At day 21, cultures were characterized histologically, biochemically, and for gene expression of vital markers. RESULTS: At day 21, 62.3% and 66.2% reduction in nitric oxide (NO) content was evident for female and male cells, respectively. G-treatment of female cells resulted in the lowest expression of nitric oxide synthase-2 (NOS2), matrix metalloproteinase-13 (MMP13), and collagen type-10 (COL10). Alpha-treatment of male cells resulted in the lowest expression of NOS2, bone morphogenic protein-2, MMP13, COL10 and tumor necrosis factor alpha induced protein-6 (TNFAIP6) relative to NC. AA and Alpha treatment resulted in the highest glycosaminoglycan (GAG) content for female and male cultures, respectively. CONCLUSION: A sex-dependent response of osteoarthritic chondrocytes to nutraceutical treatment was evident. Our results suggest the use of G for female cells and Alpha for male cells in OA applications seems to be favorable in reducing inflammation and enhancing chondrocytes' ability to form AC tissues.

In vitro effects of nutraceutical treatment on human osteoarthritic chondrocytes of females of different age and weight groups.

The in vitro effects of four nutraceuticals, catechin hydrate, gallic acid, α-tocopherol and ascorbic acid, on the ability of human osteoarthritic chondrocytes of two female obese groups to form articular cartilage (AC) tissues and to reduce inflammation were investigated. Group 1 represented thirteen females in the 50-69 years old range, an average weight of 100 kg and an average body mass index (BMI) of 34⋅06 kg/m2. Group 2 was constituted of three females in the 70-80 years old range, an average weight of 75 kg and an average BMI of 31⋅43 kg/m2. The efficacy of nutraceuticals was assessed in monolayer cultures using histological, colorimetric and mRNA gene expression analyses. AC engineered tissues of group 1 produced less total collagen and COL2A1 (38-fold), and higher COL10A1 (2⋅7-fold), MMP13 (50-fold) and NOS2 (15-fold) mRNA levels than those of group 2. In comparison, engineered tissues of group 1 had a significant decrease in NO levels from day 1 to day 21 (2⋅6-fold), as well as higher mRNA levels of FOXO1 (2-fold) and TNFAIP6 (16-fold) compared to group 2. Catechin hydrate decreased NO levels significantly in group 1 (1⋅5-fold) while increasing NO levels significantly in group 2 (3⋅8-fold). No differences from the negative control were observed in the presence of other nutraceuticals for either group. In conclusion, engineered tissues of the younger but heavier patients responded better to nutraceuticals than those from the older but leaner study participants. Finally, cells of group 2 formed better AC tissues with less inflammation and better extracellular matrix than cells of group 1.

Combining stretching and gallic acid to decrease inflammation indices and promote extracellular matrix production in osteoarthritic human articular chondrocytes.

Osteoarthritis (OA) patients undergo cartilage degradation and experience painful joint swelling. OA symptoms are caused by inflammatory molecules and the upregulation of catabolic genes leading to the breakdown of cartilage extracellular matrix (ECM). Here, we investigate the effects of gallic acid (GA) and mechanical stretching on the expression of anabolic and catabolic genes and restoring ECM production by osteoarthritic human articular chondrocytes (hAChs) cultured in monolayers. hAChs were seeded onto conventional plates or silicone chambers with or without 100 µM GA. A 5% cyclic tensile strain (CTS) was applied to the silicone chambers and the deposition of collagen and glycosaminoglycan, and gene expressions of collagen types II (COL2A1), XI (COL11A2), I (COL1A1), and X (COL10A1), and matrix metalloproteinases (MMP-1 and MMP-13) as inflammation markers, were quantified. CTS and GA acted synergistically to promote the deposition of collagen and glycosaminoglycan in the ECM by 14- and 7-fold, respectively. Furthermore, the synergistic stimuli selectively upregulated the expression of cartilage-specific proteins, COL11A2 by 7-fold, and COL2A1 by 47-fold, and, in contrast, downregulated the expression of MMP-1 by 2.5-fold and MMP-13 by 125-fold. GA supplementation with CTS is a promising approach for restoring osteoarthritic hAChs ECM production ability making them suitable for complex tissue engineering applications.

Enhanced matrix production by cocultivated human stem cells and chondrocytes under concurrent mechanical strain.

Conventional treatments of osteoarthritis have failed to re-build functional articular cartilage. Tissue engineering clinical treatments for osteoarthritis, including autologous chondrocyte implantation, provides an alternative approach by injecting a cell suspension to fill lesions within the cartilage in osteoarthritic knees. The success of chondrocyte implantation relies on the availability of chondrogenic cell lines, and their resilience to high mechanical loading. We hypothesize we can reduce the numbers of human articular chondrocytes necessary for a treatment by supplementing cultures with human adipose-derived stem cells, in which stem cells will have protective and stimulatory effects on mixed cultures when exposed to high mechanical loads, and in which coculture will enhance production of requisite extracellular matrix proteins over those produced by stretched chondrocytes alone. In this work, adipose-derived stem cells and articular chondrocytes were cultured separately or cocultivated at ratios of 3:1, 1:1, and 1:3 in static plates or under excessive cyclic tensile strain of 10% and results were compared to culturing of both cell types alone with and without cyclic strain. Results indicate 75% of chondrocytes in engineered articular cartilage can be replaced with stem cells with enhanced collagen over all culture conditions and glycosaminoglycan content over stretched cultures of chondrocytes. This can be done without observing adverse effects on cell viability. Collagen and glycosaminoglycan secretion, when compared to chondrocyte alone under 10% strain, was enhanced 6.1- and 2-fold, respectively, by chondrocytes cocultivated with stem cells at a ratio of 1:3.

The effect of hypoxia on thermosensitive poly(N-vinylcaprolactam) hydrogels with tunable mechanical integrity for cartilage tissue engineering.

Cartilage repair presents a daunting challenge in tissue engineering applications due to the low oxygen conditions (hypoxia) affiliated in diseased states. Hence, the use of biomaterial scaffolds with unique variability is imperative to treat diseased or damaged cartilage. Thermosensitive hydrogels show promise as injectable materials that can be used as tissue scaffolds for cartilage tissue regeneration. However, uses in clinical applications are limited to due mechanical stability and therapeutic efficacy to treat diseased tissue. In this study, several composite hydrogels containing poly(N-vinylcaprolactam) (PVCL) and methacrylated hyaluronic acid (meHA) were prepared using free radical polymerization to produce PVCL-graft-HA (PVCL-g-HA) and characterized using Fourier transform infrared spectroscopy, nuclear magnetic resonance, and scanning electron microscopy. Lower critical solution temperatures and gelation temperatures were confirmed in the range of 33-34°C and 41-45°C, respectively. Using dynamic sheer rheology, the temperature dependence of elastic (G') and viscous (G″) modulus between 25°C and 45°C, revealed that PVCL-g-HA hydrogels at 5% (w/v) concentration exhibited the moduli of 7 Pa (G') to 4 Pa (G″). After 10 days at 1% oxygen, collagen production on PVCL-g-HA hydrogels was 153 ± 25 µg/mg (20%) and 106 ± 18 µg/mg showing a 10-fold increase compared to meHA controls. These studies show promise in PVCL-g-HA hydrogels for the treatment of diseased or damaged articular cartilage. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1863-1873, 2017.

Synthesis of optically active helical poly(2-methoxystyrene). Enhancement of HeLa and osteoblast cell growth on optically active helical poly(2-methoxystyrene) surfaces.

Poly(2-methoxystyrene)s (P2MS) were synthesized using n-BuLi in THF and toluene at various temperatures. At -20 degrees C and higher temperatures, toluene was an effective polymerization solvent for synthesizing poly(2-methoxystyrene). Under these conditions, polymers with good yields and reasonable molecular weight distributions were obtained. Polymers synthesized under all conditions were isotactic; the most highly isotactic polymer was obtained in toluene at -20 degrees C. The m (isotactic dyad) content of the polymers synthesized in toluene at 0 degrees C and -20 degrees C was 0.65 and 0.74, respectively. Optically active helical (+) and (-) P2MS were synthesized by asymmetric polymerization utilizing (+) or (-) [2,3-dimethoxy1,4(dimethylamino)butane] (DDB)/tolyl lithium initiating complex in toluene. Asymmetric polymerizations were also carried out at 0 degrees C to synthesize optically active polymers. The optical rotations of the polymers were found to be dynamic and reversible, strongly suggesting contribution of the chiral higher ordered structure to the overall optical rotation. Geometry optimization carried out using various force fields such as MM+, AMBER and CHARMM suggests that isotactic P2MS form low energy stable helical conformations. HeLa cells showed better growth on surfaces prepared using chiral polymers compared to the surfaces prepared with achiral polymers. Similarly, chiral P2MS surfaces were also more effective as supports for mouse and human osteoblast cells. The cell attachment and growth data demonstrate that chiral P2MS surfaces were better supports compared to achiral P2MS surfaces. Atomic force microscopy (AFM) studies on surfaces prepared using chiral poly(2-methoxystyrene) showed more discrete topography features compared to surfaces prepared with achiral polymers. Thus, the surface topography may play a role in determining polymer-cell interactions.

PET imaging of soluble yttrium-86-labeled carbon nanotubes in mice.

BACKGROUND: The potential medical applications of nanomaterials are shaping the landscape of the nanobiotechnology field and driving it forward. A key factor in determining the suitability of these nanomaterials must be how they interface with biological systems. Single walled carbon nanotubes (CNT) are being investigated as platforms for the delivery of biological, radiological, and chemical payloads to target tissues. CNT are mechanically robust graphene cylinders comprised of sp(2)-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. In order to evaluate the potential usefulness of this CNT scaffold, we undertook an imaging study to determine the tissue biodistribution and pharmacokinetics of prototypical DOTA-functionalized CNT labeled with yttrium-86 and indium-111 ((86)Y-CNT and (111)In-CNT, respectively) in a mouse model. METHODOLOGY AND PRINCIPAL FINDINGS: The (86)Y-CNT construct was synthesized from amine-functionalized, water-soluble CNT by covalently attaching multiple copies of DOTA chelates and then radiolabeling with the positron-emitting metal-ion, yttrium-86. A gamma-emitting (111)In-CNT construct was similarly prepared and purified. The constructs were characterized spectroscopically, microscopically, and chromatographically. The whole-body distribution and clearance of yttrium-86 was characterized at 3 and 24 hours post-injection using positron emission tomography (PET). The yttrium-86 cleared the blood within 3 hours and distributed predominantly to the kidneys, liver, spleen and bone. Although the activity that accumulated in the kidney cleared with time, the whole-body clearance was slow. Differential uptake in these target tissues was observed following intravenous or intraperitoneal injection. CONCLUSIONS: The whole-body PET images indicated that the major sites of accumulation of activity resulting from the administration of (86)Y-CNT were the kidney, liver, spleen, and to a much less extent the bone. Blood clearance was rapid and could be beneficial in the use of short-lived radionuclides in diagnostic applications.