Objective To test the effects of sequential exposure to FGF2, 9 and 18 on human Mesenchymal Stem Cells (hMSC) differentiation during in vitro chondrogenesis. Design Control and FGF2-expanded hMSC were cultured in aggregates in the presence of rhFGF9, rhFGF18 or rhFGFR3-specific signaling FGF variants, starting at different times during the chondroinductive program. qRT-PCR and immunocytochemistry were performed at different stages. The aggregate cultures were switched to a hypertrophy-inducing medium along with rhFGFs and neutralizing antibodies against FGFR1 and FGFR3. Histological/immunohistochemical/biochemical analyses were performed. Results FGF2-exposed hMSC during expansion up-regulated Sox9 suggesting an early activation of the chondrogenic machinery. FGF2, FGF9 and 18 modulated the expression profile of FGFR1 and FGFR3 in hMSC during expansion and chondrogenesis. In combination with TGF-β, FGF9 and FGF18 inhibited chondrogenesis when added at the beginning of the program (≤d7), while exhibiting an anabolic effect when added later (≥d14), an effect mediated by FGFR3. Finally, FGFR3 signaling induced by either FGF9 or FGF18 delayed the appearance of spontaneous and induced hypertrophy-related changes. Conclusions The stage of hMSC-dependent chondrogenesis at which the growth factors are added impacts the progression of the differentiation program: increased cell proliferation and priming (FGF2); stimulated early chondrogenic differentiation (TGF-β, FGF9/FGF18) by shifting the chondrogenic program earlier; augmented ECM production (FGF9/FGF18); and delayed terminal hypertrophy (FGF9/FGF18). Collectively, these factors could be used to optimize pre-implantation conditions of hMSC when used to engineer cartilage grafts.

Directing stem cells to the heart is critical in producing an effective cell therapy for myocardial infarction (MI). Mesenchymal stem cells (MSCs) offer an exquisite drug delivery platform with environment-sensing cytokine release and MSCs have shown therapeutic potential in MI. Peptide-based targeting offers a novel method to increase cell homing, wherein MI-specific peptides, identified by phage display, are synthesized with a palmitic acid tail to facilitate cell membrane integration. Phage-peptides were screened in a mouse MI model and four peptides (CRPPR, CRKDKC, KSTRKS, and CARSKNKDC) were selected and synthesized as palmitated derivatives for further investigation. Cell coating was optimized and coating persistence and cytotoxicity were evaluated. MSCs were coated with peptides, injected into mice with MI, and MSCs in the heart quantified. Greater numbers of MSCs were found in heart of animals treated with the peptide-coated MSCs compared to uncoated controls. MSC numbers had positive correlation with MI severity in peptide-coated cells but a negative correlation in MSCs alone. A transient cell coating ("painting") method has been developed that labels cells efficiently, non-toxically and increases cell localization in MI hearts.

Background-Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.Methods-To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.Results-Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved Nglycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.Conclusion-We have identified multiple conserved amino acids within regions N-and C-terminal to the site of scission that may influence enzyme-substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.General Significance-Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.

Objective Mass exposure to alkylating agents such as nitrogen mustard (NM), whether accidental or intentional as during warfare, are known to cause systemic toxicity and severe blistering from cutaneous exposure. Thus, establishing the timing and appropriate dose of any potential drug designed to reverse or impede these toxicities is critical for wound repair and survival. Our previous data demonstrates that a single intraperitoneal injection of low-dose 25-hydroxyvitamin D3 (25(OH)D) given as early as 1 h following NM exposure is sufficient to rescue mice from pancytopenia and death. However, the duration of time following exposure where intervention is still effective as a countermeasure is unknown. In this study, we sought to assess the maximal time permissible following NM exposure where 25(OH)D still affords protection against NM-induced cutaneous injury. Additionally, we determined if a higher dose of 25(OH)D would be more efficacious at time interval where low dose 25(OH)D is no longer effective. Methods Low (5 ng) and high (50 ng) doses of 25(OH)D were administered intraperitoneally to mice following exposure to topical NM to assess wound resolution and survival. Mice were imaged and weighed daily to measure wound healing and to monitor systemic toxicity. Results We demonstrated that 5 ng 25(OH)D administered as early as 1 h and as late as 24 h post-NM exposure is able to achieve 100% recovery in mice. In contrast, intervention at and beyond 48 h of NM exposure failed to achieve full recovery and resulted in ≥60% death between days 6 and 12, demonstrating the critical nature of timely intervention with 25(OH)D at each respective dose. In order to circumvent the observed failure at >48 h exposure, we provided two consecutive doses of 5 ng or 50 ng of 25(OH)D at 48 h and 72 h post-NM exposure. Repeat dosing with 25(OH)D at 48 h and beyond led to marked improvement of lesion size with 75% recovery from mortality. Conclusions The opportunity to use 25(OH)D as a medical countermeasure for NM-induced toxicity has a finite of window for intervention. However, modifications such as repeat dosing can be an effective strategy to extend the intervention potential of 25(OH)D.

The use of sulfur mustard (SM) as a chemical weapon for warfare has once again assumed center stage, endangering civilian and the military safety. SM causes rapid local skin vesication and late-onset systemic toxicity. Most studies on SM rely on obtaining tissue and blood for characterizing burn pathogenesis and assessment of systemic pathology, respectively. However the present study focuses on developing a non-invasive method to predict mortality from high dose skin SM exposure. We demonstrate that exposure to SM leads to a dose dependent increase in wound area size on the dorsal surface of mice that is accompanied by a progressive loss in body weight loss, blood cytopenia, bone marrow destruction, and death. Thus our model utilizes local skin destruction and systemic outcome measures as variables to predict mortality in a novel skin-based model of tissue injury. Based on our recent work using vitamin D (25(OH)D) as an intervention to treat toxicity from SM-related compounds, we explored the use of 25 (OH)D in mitigating the toxic effects of SM. Here we show that 25(OH)D offers protection against SM and is the first known demonstration of an intervention that prevents SM-induced mortality. Furthermore, 25 (OH)D represents a safe, novel, and readily translatable potential countermeasure following mass toxic exposure.