Over the past several decades, there has been an ever-increasing demand for organ transplants. However, there is a severe shortage of donor organs, and as a result of the increasing demand, the gap ...between supply and demand continues to widen. A potential solution to this problem is to grow or fabricate organs using biomaterial scaffolds and a person's own cells. Although the realization of this solution has been limited, the development of new biofabrication approaches has made it more realistic. This review provides an overview of natural and synthetic biomaterials that have been used for organ/tissue development. It then discusses past and current biofabrication techniques, with a brief explanation of the state of the art. Finally, the review highlights the need for combining vascularization strategies with current biofabrication techniques. Given the multitude of applications of biofabrication technologies, from organ/tissue development to drug discovery/screening to development of complex in vitro models of human diseases, these manufacturing technologies can have a significant impact on the future of medicine and health care.
Materials that support the assembly of new vasculature are critical for regenerative medicine. Controlling the scaffold’s mechanical properties may help to optimize neovascularization within ...implanted biomaterials. However, reducing the stiffness of synthetic hydrogels usually requires decreasing polymer densities or increasing chain lengths, both of which accelerate degradation. We synthesized enzymatically-degradable poly(ethylene glycol) hydrogels with compressive moduli from 2 to 18 kPa at constant polymer density, chain length, and proteolytic degradability by inserting an allyloxycarbonyl functionality into the polymer backbone. This group competes with acrylates during photopolymerization to alter the crosslink network structure and reduce the hydrogel’s stiffness. Hydrogels that incorporated (soft) or lacked (stiff) this group were implanted subcutaneously in rats to investigate the role of stiffness on host tissue interactions. Changes in tissue integration were quantified after 4 weeks
via
the hydrogel area replaced by native tissue (tissue area fraction), yielding 0.136 for softer vs. 0.062 for stiffer hydrogels. Including soluble FGF-2 and PDGF-BB improved these responses to 0.164 and 0.089, respectively. Softer gels exhibited greater vascularization with 8.6 microvessels mm
−2
compared to stiffer gels at 2.4 microvessels mm
−2
. Growth factors improved this to 11.2 and 4.9 microvessels mm
−2
, respectively. Softer hydrogels tended to display more sustained responses, promoting neovascularization and tissue integration in synthetic scaffolds.
Surgical delay is a well-described technique to improve survival of random and pedicled cutaneous flaps. The aim of this study was to test the topical agents minoxidil and iloprost as agents of ...pharmacologic delay to induce vascular remodeling and decrease overall flap necrosis as an alternative to surgical delay.
Seven groups were studied (n = 8 in each group), including the following: vehicle, iloprost, or minoxidil before treatment only; vehicle, iloprost, or minoxidil before and after treatment; and a standard surgical delay group as a positive control. Surgical flaps (caudally based modified McFarlane myocutaneous skin flaps) were elevated after 14 days of pretreatment, reinset isotopically, and observed at various time points until postoperative day 7. Gross viability, histology, Doppler blood flow, perfusion imaging, tissue oxygenation measurement, and vascular casting were performed for analysis.
Pharmacologic delay with preoperative application of topical minoxidil or iloprost was found to have comparable flap viability when compared to surgical delay. Significantly increased viability in all treatment groups was observed when compared with vehicle. Continued postoperative treatment with topical agents had no effect on flap viability. The mechanism of improved flap viability was inducible increases in flap blood volume and perfusion rather than the acute vasodilatory effects of the topical agents or decreased flap hypoxia.
Preoperative topical application of the vasodilators minoxidil or iloprost improved flap viability comparably to surgical delay. Noninvasive pharmacologic delay may reduce postoperative complications without the need for an additional operation.
Preoperative use of topical vasodilators may lead to improved flap viability without the need for a surgical delay procedure. This study may inform future clinical trials examining utility of preoperative topical vasodilators in flap surgery.
•Obstacles for CED of CAR T cells are sedimentation, tube clogging, low viability.•LV Hydrogel is a biodegradable hyaluronic acid based biomaterial.•LV Hydrogel provides 20-fold increase in the ...delivery rate of CAR T cells.•Migration capacity and cytotoxicity of CAR T cells remain the same after infusion.•LV hydrogel has no toxic effect on rodent brain and fully disintegrated after 2 weeks.
Convection Enhanced Delivery (CED) infuses therapeutic agents directly into the intracranial area continuously under pressure. The convection improves the distribution of therapeutics such as those aimed at brain tumors. Although CED successfully delivers small therapeutic agents, this technique fails to effectively deliver cells largely due to cell sedimentation during delivery. To overcome this limitation, we have developed a low viscosity hydrogel (LVHydrogel), which is capable of retaining cells in suspension. In this study, we evaluated whether LVHydrogel can effectively act as a carrier for the CED of tumor-specific chimeric antigen receptor (CAR) T cells. CAR T cells were resuspended in saline or LVHydrogel carriers, loaded into syringes, and passed through the CED system for 5 h. CAR T cells submitted to CED were counted and the efficiency of delivery was determined. In addition to delivery, the ability of CAR T cells to migrate and induce cytotoxicity was evaluated. Our studies demonstrate that LVHydrogel is a superior carrier for CED in comparison to saline. The efficiency of cell delivery in saline carrier was only ∼3–5% of the total cells whereas delivery by the LVHydrogel carrier was much higher, reaching ∼45–75%. Migration and Cytotoxicity was similar in both carriers in non-infused samples but we found superior cytotoxicity in LVHydrogel group post-infusion. We demonstrate that LVHydrogel, a biodegradable biomaterial which does not cause acute toxicity on preclinical animal models, prevents cellular sedimentation during CED and presents itself as a superior carrier to the current carrier, saline, for the CED of CAR T cells.
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•Assessed porphyrin-containing biodegradable electrospun dissolved oxygen (DO) sensors.•Red excitation and near-infrared emission ideal for in vivo applications.•In situ setup for ...phosphorescent lifetime collection and real-time DO verification.•Sensors exhibit linear Stern-Volmer plots for ease of calibration and interpretation.•Performance of core-shell and blended PCL:gelatin fibers similar to pure PCL sensor.
The ability to continuously monitor tissue oxygenation in vivo is highly desired for a variety of biomedical applications. Implantable optical oxygen sensors can be constructed through incorporation of an oxygen-sensitive chromophore within a biocompatible polymer host. In this work, a Pd (II) benzoporphyrin that absorbs (630 nm) and phosphoresces (810 nm) within the optical tissue window was incorporated into various biodegradable polymer matrices via electrospinning. A unique in situ fluorimeter setup was constructed and used to rigorously characterize the sensor performance in the physiologically relevant dissolved oxygen range (∼0−90 μM) in 37 °C phosphate buffered saline. Single-component polycaprolactone (PCL) fibers exhibited high sensitivity (KSV: 1.25 × 105 M−1) and monoexponential decay curves, indicating a homogenous chromophore environment. The ability to move from a single-component composition (PCL) to blended (PCL:gelatin) and core-shell systems (PCL:gelatin-PCL) without compromising the desirable properties of the single-component sensor was demonstrated. These variations may offer the potential to tune degradation time of the resulting sensor while maintaining high sensitivity and monoexponential decays. In contrast, blended 50:50 PCL:poly(d,l-lactide-co-glycolide) (PLGA) exhibited significantly reduced sensitivity (KSV: 6.66 × 104 M−1) and biexponential decays. Despite the observation of biexponential lifetime decays for PCL:PLGA, all electrospun sensors exhibited linear Stern-Volmer behavior over the physiologically relevant dissolved oxygen range. This work rigorously evaluated biodegradable candidates for in vivo oxygen sensing under physiologically relevant conditions in which the character of the individual phosphorescence decay curves offered insight into the nature of the chromophore environment.
The effects of mechanical cues on cell behaviors in 3D remain difficult to characterize as the ability to tune hydrogel mechanics often requires changes in the polymer density, potentially altering ...the material’s biochemical and physical characteristics. Additionally, with most PEG diacrylate (PEGDA) hydrogels, forming materials with compressive moduli less than ∼10 kPa has been virtually impossible. Here, we present a new method of controlling the mechanical properties of PEGDA hydrogels independent of polymer chain density through the incorporation of additional vinyl group moieties that interfere with the cross-linking of the network. This modification can tune hydrogel mechanics in a concentration dependent manner from <1 to 17 kPa, a more physiologically relevant range than previously possible with PEG-based hydrogels, without altering the hydrogel’s degradation and permeability. Across this range of mechanical properties, endothelial cells (ECs) encapsulated within MMP-2/MMP-9 degradable hydrogels with RGDS adhesive peptides revealed increased cell spreading as hydrogel stiffness decreased in contrast to behavior typically observed for cells on 2D surfaces. EC-pericyte cocultures exhibited vessel-like networks within 3 days in highly compliant hydrogels as compared to a week in stiffer hydrogels. These vessel networks persisted for at least 4 weeks and deposited laminin and collagen IV perivascularly. These results indicate that EC morphogenesis can be regulated using mechanical cues in 3D. Furthermore, controlling hydrogel compliance independent of density allows for the attainment of highly compliant mechanical regimes in materials that can act as customizable cell microenvironments.
DNA‐based erasers: The designed strand‐displacement reactions between dynamic DNA probes (carrying fluorescent dyes (★) and quencher domains (•)) and DNA–antibody conjugates generate detachable ...immunofluoresence‐reporting complexes that can be assembled (first image) and disassembled (second image) reversibly within fixed cells. With this system, different protein targets can be imaged sequentially within the same cells (third image).
OBJECTIVES:
Acute spinal cord injury is a devastating injury that may lead to loss of independent function. Stem-cell therapies have shown promise; however, a clinically efficacious stem-cell therapy ...has yet to be developed. Functionally, endothelial progenitor cells induce angiogenesis, and neural stem cells induce neurogenesis. In this study, we explored using a multimodal therapy combining endothelial progenitor cells with neural stem cells encapsulated in a bioactive biomimetic hydrogel matrix to facilitate stem cell–induced neurogenesis and angiogenesis in a rat hemisection spinal cord injury model.
DESIGN:
Laboratory experimentation.
SETTING:
University laboratory.
SUBJECTS:
Female Fischer 344 rats.
INTERVENTIONS:
Three groups of rats: 1) control, 2) biomimetic hydrogel therapy, and 3) combined neural stem cell, endothelial progenitor cell, biomimetic hydrogel therapy underwent right-sided spinal cord hemisection at T9–T10. The blinded Basso, Beattie, and Bresnahan motor score was obtained weekly; after 4 weeks, observational histologic analysis of the injured spinal cords was completed.
MEASUREMENTS AND MAIN RESULTS:
Blinded Basso, Beattie, and Bresnahan motor score of the hind limb revealed significantly improved motor function in rats treated with combined neural stem cell, endothelial progenitor cell, and biomimetic hydrogel therapy (
p
< 0.05) compared with the control group. The acellular biomimetic hydrogel group did not demonstrate a significant improvement in motor function compared with the control group. Immunohistochemistry evaluation of the injured spinal cords demonstrated de novo neurogenesis and angiogenesis in the combined neural stem cell, endothelial progenitor cell, and biomimetic hydrogel therapy group, whereas, in the control group, a gap or scar was found in the injured spinal cord.
CONCLUSIONS:
This study demonstrates proof of concept that multimodal therapy with endothelial progenitor cells and neural stem cells combined with a bioactive biomimetic hydrogel can be used to induce de novo CNS tissue in an injured rat spinal cord.
The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules ...that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells.
A class of reactive DNA circuits was adapted as erasable molecular imaging probes that allow fluorescent reporting complexes to be assembled and disassembled on a biological specimen. Circuit ...reactions are sequence-dependent and therefore facilitate multiplexed (multicolor) detection. Yet, the ability to disassemble reporting complexes also allows fluorophores to be removed and new circuit complexes to be used to label additional markers. Thus, these probes present opportunities to increase the total number of molecular targets that can be visualized on a biological sample by allowing multiple rounds of fluorescence microscopy to be performed.
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