Chitosan coatings with distinct innate immune bioactivities differentially stimulate angiogenesis, osteogenesis and chondrogenesis in poly-caprolactone scaffolds with controlled interconnecting pore size.

This study tested whether osseous integration into poly (ε-caprolactone) (PCL) bioplastic scaffolds with fully-interconnecting 155 ± 8 µm pores is enhanced by an adhesive, non-inflammatory 99% degree of deacetylation (DDA) chitosan coating (99-PCL), or further incorporation of pro-inflammatory 83% DDA chitosan microparticles (83-99-PCL) to accelerate angiogenesis. New Zealand White rabbit osteochondral knee defects were press-fit with PCL, 99-PCL, 83-99-PCL, or allowed to bleed (drill-only). Between day 1 and 6 weeks of repair, drill-only defects repaired by endochondral ossification, with an 8-fold higher bone volume fraction (BVF) versus initial defects, compared to a 2-fold (99-PCL), 1.1-fold (PCL), or 0.4-fold (83-99-PCL) change in BVF. Hematoma innate immune cells swarmed to 83-99-PCL, elicited angiogenesis throughout the pores and induced slight bone resorption. PCL and 99-PCL pores were variably filled with cartilage or avascular mesenchyme near the bone plate, or angiogenic mesenchyme into which repairing trabecular bone infiltrated up to 1 mm deep. More repair cartilage covered the 99-PCL scaffold (65%) than PCL (18%) or 83-99-PCL (0%) (p < 0.005). We report the novel finding that non-inflammatory chitosan coatings promoted cartilage infiltration into and over a bioplastic scaffold, and were compatible with trabecular bone integration. This study also revealed that in vitro osteogenesis assays have limited ability to predict osseous integration into porous scaffolds, because (1) in vivo, woven bone integrates from the leading edge of regenerating trabecular bone and not from mesenchymal cells adhering to scaffold surfaces, and (2) bioactive coatings that attract inflammatory cells induce bone resorption.

Cationic osteogenic peptide P15-CSP coatings promote 3-D osteogenesis in poly(epsilon-caprolactone) scaffolds of distinct pore size.

P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ɛ-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained nonmineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, fourfold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p < 0.05). 3-D Micro-CT revealed asymmetric mineral deposition consistent with histological calcium staining. This study provides proof-of-concept that P15-CSP coatings are osteoconductive in PCL pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2171-2181, 2017.

Prolactin promotes cartilage survival and attenuates inflammation in inflammatory arthritis.

Chondrocytes are the only cells in cartilage, and their death by apoptosis contributes to cartilage loss in inflammatory joint diseases, such as rheumatoid arthritis (RA). A putative therapeutic intervention for RA is the inhibition of apoptosis-mediated cartilage degradation. The hormone prolactin (PRL) frequently increases in the circulation of patients with RA, but the role of hyperprolactinemia in disease activity is unclear. Here, we demonstrate that PRL inhibits the apoptosis of cultured chondrocytes in response to a mixture of proinflammatory cytokines (TNF-α, IL-1ß, and IFN-γ) by preventing the induction of p53 and decreasing the BAX/BCL-2 ratio through a NO-independent, JAK2/STAT3-dependent pathway. Local treatment with PRL or increasing PRL circulating levels also prevented chondrocyte apoptosis evoked by injecting cytokines into the knee joints of rats, whereas the proapoptotic effect of cytokines was enhanced in PRL receptor-null (Prlr(-/-)) mice. Moreover, eliciting hyperprolactinemia in rats before or after inducing the adjuvant model of inflammatory arthritis reduced chondrocyte apoptosis, proinflammatory cytokine expression, pannus formation, bone erosion, joint swelling, and pain. These results reveal the protective effect of PRL against inflammation-induced chondrocyte apoptosis and the therapeutic potential of hyperprolactinemia to reduce permanent joint damage and inflammation in RA.

Subchondral pre-solidified chitosan/blood implants elicit reproducible early osteochondral wound-repair responses including neutrophil and stromal cell chemotaxis, bone resorption and repair, enhanced repair tissue integration and delayed matrix deposition.

BACKGROUND: In this study we evaluated a novel approach to guide the bone marrow-driven articular cartilage repair response in skeletally aged rabbits. We hypothesized that dispersed chitosan particles implanted close to the bone marrow degrade in situ in a molecular mass-dependent manner, and attract more stromal cells to the site in aged rabbits compared to the blood clot in untreated controls. METHODS: Three microdrill hole defects, 1.4 mm diameter and 2 mm deep, were created in both knee trochlea of 30 month-old New Zealand White rabbits. Each of 3 isotonic chitosan solutions (150, 40, 10 kDa, 80% degree of deaceylation, with fluorescent chitosan tracer) was mixed with autologous rabbit whole blood, clotted with tissue factor to form cylindrical implants, and press-fit in drill holes in the left knee while contralateral holes received tissue factor or no treatment. At day 1 or day 21 post-operative, defects were analyzed by micro-computed tomography, histomorphometry and stereology for bone and soft tissue repair. RESULTS: All 3 implants filled the top of defects at day 1 and were partly degraded in situ at 21 days post-operative. All implants attracted neutrophils, osteoclasts and abundant bone marrow-derived stromal cells, stimulated bone resorption followed by new woven bone repair (bone remodeling) and promoted repair tissue-bone integration. 150 kDa chitosan implant was less degraded, and elicited more apoptotic neutrophils and bone resorption than 10 kDa chitosan implant. Drilled controls elicited a poorly integrated fibrous or fibrocartilaginous tissue. CONCLUSIONS: Pre-solidified implants elicit stromal cells and vigorous bone plate remodeling through a phase involving neutrophil chemotaxis. Pre-solidified chitosan implants are tunable by molecular mass, and could be beneficial for augmented marrow stimulation therapy if the recruited stromal cells can progress to bone and cartilage repair.

Chitosan rate of uptake in HEK293 cells is influenced by soluble versus microparticle state and enhanced by serum-induced cell metabolism and lactate-based media acidification.

UNLABELLED: Chitosan is a biocompatible polysaccharide composed of glucosamine and N-acetylglucosamine. The polymer has a unique behavior of fluctuating between soluble chains at pH 6 and insoluble microparticles at pH 7. The purpose of this study was to test the hypothesis that chitosan structure, solubility state, and serum influence the rate of cell uptake. Chitosans with 80% and 95% degree of deacetylation (medium and low viscosity) were tagged with rhodamine and analyzed for particle size, media solubility, and uptake by HEK293 epithelial cells using live confocal microscopy and flow cytometry. In media pH 7.4 with or without 10% serum, chitosans fully precipitated into 0.5 to 1.4 µm diameter microparticles with a slight negative charge. During 24 h of culture in serum-free medium, chitosan particles remained extracellular. In cultures with serum, particles were taken up into intracellular vesicles in a serum dose-dependent manner. Opsonization of chitosan with serum, or replacement of serum by epidermal growth factor (EGF) failed to mediate serum-free chitosan particle uptake. Serum stimulated cells to acidify the media, partly by lactate generation. Media acidified to pH 6.5 by 7 mM lactate maintained 50% of chitosan in the soluble fraction, and led to minor uniform serum-free uptake in small vesicles. CONCLUSION: Media acidification mediates minor in vitro uptake of non-biofouled soluble chitosan chains, while serum-biofouled insoluble chitosan microparticles require sustained serum exposure to generate energy required for macropinocytosis.

The cartilage-bone interface.

In the knee joint, the purpose of the cartilage-bone interface is to maintain structural integrity of the osteochondral unit during walking, kneeling, pivoting, and jumping--during which tensile, compressive, and shear forces are transmitted from the viscoelastic articular cartilage layer to the much stiffer mineralized end of the long bone. Mature articular cartilage is integrated with subchondral bone through a approximately 20 to approximately 250 microm thick layer of calcified cartilage. Inside the calcified cartilage layer, perpendicular chondrocyte-derived collagen type II fibers become structurally cemented to collagen type I osteoid deposited by osteoblasts. The mature mineralization front is delineated by a thin approximately 5 microm undulating tidemark structure that forms at the base of articular cartilage. Growth plate cartilage is anchored to epiphyseal bone, sometimes via a thin layer of calcified cartilage and tidemark, while the hypertrophic edge does not form a tidemark and undergoes continual vascular invasion and endochondral ossification (EO) until skeletal maturity upon which the growth plates are fully resorbed and replaced by bone. In this review, the formation of the cartilage-bone interface during skeletal development and cartilage repair, and its structure and composition are presented. Animal models and human anatomical studies show that the tidemark is a dynamic structure that forms within a purely collagen type II-positive and collagen type I-negative hyaline cartilage matrix. Cartilage repair strategies that elicit fibrocartilage, a mixture of collagen type I and type II, are predicted to show little tidemark/calcified cartilage regeneration and to develop a less stable repair tissue-bone interface. The tidemark can be regenerated through a bone marrow-driven growth process of EO near the articular surface.

Biodegradable chitosan particles induce chemokine release and negligible arginase-1 activity compared to IL-4 in murine bone marrow-derived macrophages.

Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear.In vitro, macrophages stimulated with high doses of chitosan (≥ 500 µg/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5-500 µg/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-γ/LPS (classical activation), 1 µm diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure, BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-γ/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1ß, TNF-α, MIP-1α/MIP-1ß), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1α/ß, GM-CSF, RANTES, IL-1ß), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms.

Effect of chitosan particles and dexamethasone on human bone marrow stromal cell osteogenesis and angiogenic factor secretion.

Chitosan is a polysaccharide scaffold used to enhance cartilage repair during treatments involving bone marrow stimulation, and it is reported to increase angiogenesis and osteogenesis in vivo. Here, we tested the hypotheses that addition of chitosan particles to the media of human bone marrow stromal cell (BMSC) cultures stimulates osteogenesis by promoting osteoblastic differentiation and by favoring the release of angiogenic factors in vitro. Confluent BMSCs were cultured for 3 weeks with 16% fetal bovine serum, ascorbate-2-phosphate and disodium beta-glycerol phosphate, in the absence or presence of dexamethasone, an anti-inflammatory glucocorticoid commonly used as an inducer of BMSC osteoblast differentiation in vitro. As expected, dexamethasone slowed cell division, stimulated alkaline phosphatase activity and enhanced matrix mineralization. Added chitosan particles accumulated intra- and extracellularly and, while not affecting most osteogenic features, they inhibited osteocalcin release to the media at day 14 and interfered with mineralized matrix deposition. Interestingly, dexamethasone promoted cell attachment and suppressed the release and activation of matrix metalloprotease-2 (MMP-2). While chitosan particles had no effect on the release of angiogenic factors, dexamethasone significantly inhibited (p<0.05 to p<0.0001) the release of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukins 1beta, 4, 6, and 10 (IL-1beta, IL-4, IL-6, IL-10), and a host of other inflammatory factors that were constitutively secreted by BMSCs. These results demonstrate that chitosan particles alone are not sufficient to promote osteoblast differentiation of BMSCs in vitro, and suggest that chitosan promotes osteogenesis in vivo through indirect mechanisms. Our data further show that continuous addition of dexamethasone promotes osteoblastic differentiation in vitro partly by inhibiting gelatinase activity and by suppressing inflammatory cytokines which result in increased cell attachment and cell cycle exit.

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin.

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.