Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20 days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8 days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. Statement of Significance Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.
Fibrin is an attractive material for regenerative medicine applications. It not only forms a polymer but also contains cryptic matrikines that are released upon its activation/degradation and enhance the regenerative process. Despite this advantageous biology associated with fibrin, commercially available systems (e.g. TISSEEL) display limited regenerative capacity. This limitation is in part due to formulations that are optimized for tissue sealant applications and result in dense fibrous networks that limit cell infiltration. Recent evidence suggests that polymerization knob 'B' engagement of polymerization hole 'b' activates an alternative polymerization mechanism in fibrin, which may result in altered single fiber mechanical properties. We hypothesized that augmenting fibrin polymerization through the addition of PEGylated knob peptides with specificity to hole 'b' (AHRPYAAC-PEG) would result in distinct fibrin polymer architectures with grossly different physical properties. Polymerization dynamics, polymer architecture, diffusivity, viscoelasticity, and degradation dynamics were analyzed. Results indicate that specific engagement of hole 'b' with PEGylated knob 'B' conjugates during polymerization significantly enhances the porosity of and subsequent diffusivity through fibrin polymers. Paradoxically, these polymers also display increased viscoelastic properties and decreased susceptibility to degradation. As a result, fibrin polymer strength was significantly augmented without any adverse effects on angiogenesis within the modified polymers.
Carbohydrates and glycoconjugates have been shown to exert pro-inflammatory effects on the dendritic cells (DCs), supporting pathogen-induced innate immunity and antigen processing, as well as immunosuppressive effects in the tolerance to self-proteins. Additionally, the innate inflammatory response to implanted biomaterials has been hypothesized to be mediated by inflammatory cells interacting with adsorbed proteins, many of which are glycosylated. However, the molecular factors relevant for surface displayed glycoconjugate modulation of dendritic cell (DC) phenotype are unknown. Thus, in this study, a model system was developed to establish the role of glycan composition, density, and carrier cationization state on DC response. Thiol modified glycans were covalently bound to a model protein carrier, maleimide functionalized bovine serum albumin (BSA), and the number of glycans per BSA modulated. Additionally, the carrier isoelectric point was scaled from a pI of ~4.0 to ~10.0 using ethylenediamine (EDA). The DC response to the neoglycoconjugates adsorbed to wells of a 384-well plate was determined via a high throughput assay. The underlying trends in DC phenotype in relation to conjugate properties were elucidated via multivariate general linear models. It was found that glycoconjugates with more than 20 glycans per carrier had the greatest impact on the pro-inflammatory response from DCs, followed by conjugates having an isoelectric point above 9.5. Surfaces displaying terminal α1-2 linked mannose structures were able to increase the inflammatory DC response to a greater extent than did any other terminal glycan structure. The results herein can be applied to inform the design of the next generation of combination products and biomaterials for use in future vaccines and implanted materials.
HIV patients on highly active antiretroviral therapy (HAART) exhibit elevated incidence of cardiovascular disease (CVD), including a higher risk of myocardial infarction and prevalence of atherosclerotic lesions, as well as increases in markers of subclinical atherosclerosis including increased carotid artery intima-media thickness (c-IMT), increased arterial stiffness, and impaired flow-mediated dilation. Both HAART and HIV-infection are independent risk factors for atherosclerosis and myocardial infarction. Studies implicate the HIV proteins tat, gp120, vpu, and nef in early on-set atherosclerosis. The objective of this study was to quantify the role of expression of HIV-1 proteins on the vascular function, biomechanics, and geometry of common carotid arteries and aortas. This study employed NL4-3Δ gag/pol transgenic mice (HIV-Tg), which contain the genetic sequence for the HIV-1 proteins env, tat, nef, rev, vif, vpr, and vpu but lacks the gag and pol genes and reports that HIV-Tg mice have impaired aortic endothelial function, increased c-IMT, and increased arterial stiffness. Further, HIV-Tg arteries show decreased elastin content, increased cathepsin K and cathepsin S activity, and increased mechanical residual stress. Thus, mice that express HIV proteins exhibit pre-clinical markers of atherosclerosis and these markers correlate with changes in markers of vascular remodeling. These findings are consistent with the hypothesis that HIV-proteins, independent of HAART treatment or HIV infection, could play a role in of the development of CVD.
HIV positive patients on highly active antiretroviral therapy (HAART) have shown elevated incidence of a number of non-AIDS defining co-morbidities, including cardiovascular disease. Given that HAART regimens contain a combination of at least three drugs, that disease management often requires adjustment of these regimens, and HIV, independent of HAART, also plays a role in development of co-morbidities, determining the role of specific HAART drugs and HIV infection itself from clinical data remains challenging. To characterize specific mediators and underlying mechanisms of disease, in vitro and in vivo animal models are required, in parallel with clinical data. Given its low cost azidothymidine (AZT) contributes to the backbone of a large proportion of HAART treated patients in the developing world where much of the global burden of HIV resides. The goal of this study was to test the hypothesis that AZT can lead to proatherogenic changes including the subclinical markers of arterial stiffening and intima-media thickening in mice. AZT (100. mg/kg) or vehicle was administered to wild-type FVB/N mice via oral gavage for 35 days. Cylindrical biaxial biomechanical tests on the common carotid arteries and suprarenal aortas exhibited arterial stiffening in AZT mice compared to controls. Multiphoton microscopy and histology demonstrated that AZT led to increased intima-media thickness. These data correlated with decreased elastin content and increased protease activity as measured by cathepsin zymography; no differences were observed in collagen content or organization, in vivo axial stretch, or opening angle. Thus, this study suggests the drug AZT has significant effects on the development of subclinical markers of atherosclerosis.
Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained invivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.
Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press This study aimed to determine whether autologous orthobiologic tissue source affects pain and functional outcomes in patients with symptomatic knee osteoarthritis (OA) who received microfragmented adipose tissue (MFAT) or bone marrow aspirate concentrate (BMAC) injection. We retrospectively reviewed prospectively collected data from patients who received BMAC or MFAT injection for symptomatic knee OA. Patients completed baseline and follow-up surveys. Each survey included the Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire, Emory Quality of Life (EQOL) questionnaire, and Visual Analog Scale (VAS) for pain. The follow-up responses were compared with baseline for all patients and between BMAC and MFAT groups. A total of 110 patients met inclusion criteria, with 76 patients (BMAC 41, MFAT 35) and 106 knees (BMAC 58, MFAT 48) having appropriate follow-up data. The BMAC group included 17 females and 24 males, with a mean age of 59 ± 11 years. The MFAT group included 23 females and 12 males, with a mean age of 63 ± 11 years. Minimum follow-up time was 0.5 years. Mean follow-up time was 1.80 ± 0.88 years for BMAC and 1.09 ± 0.49 years for MFAT. Both groups had significant improvement in EQOL, VAS, and all KOOS parameters preprocedure versus postprocedure (p <.001). There was not a significant difference when comparing postprocedure scores between groups (p =.09,.38,.63,.94,.17,.15,.70, respectively). These data demonstrate significant improvement in pain and function with both MFAT and BMAC injections in patients with symptomatic knee OA without a significant difference in improvement when comparing the two autologous tissue sources. Stem Cells Translational Medicine 2019;8:1149–1156.
Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4<sup>+</sup> T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.
High-dose-rate (HDR) brachytherapy has become a popular treatment modality for localized prostate cancer. Prostate HDR treatment involves placing 10 to 20 catheters (needles) into the prostate gland, and then delivering radiation dose to the cancerous regions through these catheters. These catheters are often inserted with transrectal ultrasound (TRUS) guidance and the HDR treatment plan is based on the CT images. The main challenge for CT-based HDR planning is to accurately segment prostate volume in CT images due to the poor soft tissue contrast and additional artifacts introduced by the catheters. To overcome these limitations, we propose a novel approach to segment the prostate in CT images through TRUS-CT deformable registration based on the catheter locations. In this approach, the HDR catheters are reconstructed from the intra-operative TRUS and planning CT images, and then used as landmarks for the TRUS-CT image registration. The prostate contour generated from the TRUS images captured during the ultrasound-guided HDR procedure was used to segment the prostate on the CT images through deformable registration. We conducted two studies. A prostate-phantom study demonstrated a submillimeter accuracy of our method. A pilot study of 5 prostate-cancer patients was conducted to further test its clinical feasibility. All patients had 3 gold markers implanted in the prostate that were used to evaluate the registration accuracy, as well as previous diagnostic MR images that were used as the gold standard to assess the prostate segmentation. For the 5 patients, the mean gold-marker displacement was 1.2 mm; the prostate volume difference between our approach and the MRI was 7.2%, and the Dice volume overlap was over 91%. Our proposed method could improve prostate delineation, enable accurate dose planning and delivery, and potentially enhance prostate HDR treatment outcome.
To conduct a patient-specific computational modeling of the aortic valve, 3-D aortic valve anatomic geometries of an individual patient need to be reconstructed from clinical 3-D cardiac images. Currently, most of computational studies involve manual heart valve geometry reconstruction and manual finite element (FE) model generation, which is both time-consuming and prone to human errors. A seamless computational modeling framework, which can automate this process based on machine learning algorithms, is desirable, as it can not only eliminate human errors and ensure the consistency of the modeling results but also allow fast feedback to clinicians and permits a future population-based probabilistic analysis of large patient cohorts. In this study, we developed a novel computational modeling method to automatically reconstruct the 3-D geometries of the aortic valve from computed tomographic images. The reconstructed valve geometries have built-in mesh correspondence, which bridges harmonically for the consequent FE modeling. The proposed method was evaluated by comparing the reconstructed geometries from 10 patients with those manually created by human experts, and a mean discrepancy of 0.69 mm was obtained. Based on these reconstructed geometries, FE models of valve leaflets were developed, and aortic valve closure from end systole to middiastole was simulated for 7 patients and validated by comparing the deformed geometries with those manually created by human experts, and a mean discrepancy of 1.57 mm was obtained. The proposed method offers great potential to streamline the computational modeling process and enables the development of a preoperative planning system for aortic valve disease diagnosis and treatment.