Cell Delivery Platform Research Articles

Encapsulation of human natural killer cells into novel gelatin-based polymeric hydrogel networks.

In the innate immune system, natural killer (NK) cells are effector lymphocytes which control several tumor types and microbial infections by limiting disease spread and tissue damage. With tumor cell killing abilities, with no priming or prior activation, NKs are potential anti-cancer therapies. In clinical practice, NKs are used in intravenous injections as they typically grow as suspension, similar to other blood cells. In this study, we designed a novel and effective biomaterial-based platform for NK cell delivery, which included in-situ NK cell encapsulation into three-dimensional (3D) biocompatible polymeric scaffolds for potential anti-cancer treatments. Depending on physical cross-linking between an alginate (ALG) polymer and a divalent cation, two natural polymers (gelatin (GEL) and hyaluronic acid (HA)) penetrated into pores and generated an inter-penetrating hydrogel system with improved mechanical properties and stability. After extensive characterization of hydrogels, NK cells were encapsulated inside using our in-situ gelation procedure to provide a biomimetic microenvironment.&#xD.

Anti-Stress and Anti-ROS Effects of MnOx-Functionalized Thermosensitive Nanohydrogel Protect BMSCs for Intervertebral Disc Degeneration Repair.

Stem cell transplantation is proven to be a promising strategy for intervertebral disc degeneration (IDD) repair. However, replicative senescence of bone marrow-derived mesenchymal stem cells (BMSCs), shear damage during direct injection, mechanical stress, and the reactive oxygen species (ROS)-rich microenvironment in degenerative intervertebral discs (IVDs) cause significant cellular damage and limit the therapeutic efficacy. Here, an injectable manganese oxide (MnOx)-functionalized thermosensitive nanohydrogel is proposed for BMSC transplantation for IDD therapy. The MnOx-functionalized thermosensitive nanohydrogel not only successfully protects BMSCs from shear force and mechanical stress before and after injection, but also repairs the harsh high-ROS environment in degenerative IVDs, thus effectively increasing the viability of BMSCs and resident nucleus pulposus cells (NPCs). The MnOx-functionalized thermosensitive nanohydrogel provides mechanical protection for stem cells and helps to remove endogenous ROS, providing a promising stem cell delivery platform for the treatment of IDD.

Development of a biocompatible 3D hydrogel scaffold using continuous liquid interface production for the delivery of cell therapies to treat recurrent glioblastoma

AbstractGlioblastoma (GBM) is the most common primary malignant brain tumor diagnosed in adults, carrying with it an extremely poor prognosis and limited options for effective treatment. Various cell therapies have emerged as promising candidates for GBM treatment but fail in the clinic due to poor tumor trafficking, poor transplantation efficiency, and high systemic toxicity. In this study, we design, characterize, and test a 3D‐printed cell delivery platform that can enhance the survival of therapeutic cells implanted in the GBM resection cavity. Using continuous liquid interface production (CLIP) to generate a biocompatible 3D hydrogel, we demonstrate that we can effectively seed neural stem cells (NSCs) onto the surface of the hydrogel, and that the cells can proliferate to high densities when cultured for 14 days in vitro. We show that NSCs seeded on CLIP scaffolds persist longer than freely injected cells in vivo, proliferating to 20% higher than their original density in 6 days after implantation. Finally, we demonstrate that therapeutic fibroblasts seeded on CLIP more effectively suppress tumor growth and extend survival in a mouse model of LN229 GBM resection compared to the scaffold or therapeutic cells alone. These promising results demonstrate the potential to leverage CLIP to design hydrogels with various features to control the delivery of different types of cell therapies. Future work will include a more thorough evaluation of the immunological response to the material and improvement of the printing resolution for biocompatible aqueous resins.

3D Porous Polymer Scaffold-Conjugated KGF-Mimetic Peptide Promotes Functional Skin Regeneration in Chronic Diabetic Wounds.

The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.

Fabrication and Characterization of Collagen–Magnetic Particle Composite Microbeads for Targeted Cell Adhesion and Proliferation

The integration of the biocompatibility of collagen and the remote-control ability of magnetic elements serves as both a cell scaffold and an actuator. We studied the preparation, characterization, and potential applications of collagen–magnetic particle composite microbeads (CMPMBs). The interplay among collagen concentration, particle size, and surface roughness was found to influence cell adhesion and proliferation. Adsorption and desorption tests showed the reversible attachment of the particles to magnetic sheets, enabling precise spatial control and targeted cell delivery. The particles demonstrated their utility as cell carriers, supporting cell migration and proliferation. These findings showcase the potential of CMPMBs as a promising platform for advanced cell delivery and tissue regeneration applications. The ability to fine-tune particle properties and manipulate them using magnetic fields offers new possibilities for creating complex tissue constructs and controlling cellular behavior, which could contribute to the development of more effective regenerative therapies and tissue engineering approaches.

Modeling of a Bioengineered Immunomodulating Microenvironment for Cell Therapy.

Cell delivery and encapsulation platforms are under development for the treatment of Type 1 Diabetes among other diseases. For effective cell engraftment, these platforms require establishing an immune-protected microenvironment as well as adequate vascularization and oxygen supply to meet the metabolic demands of the therapeutic cells. Current platforms rely on 1) immune isolating barriers and indirect vascularization or 2) direct vascularization with local or systemic delivery of immune modulatory molecules. Supported by experimental data, here a broadly applicable predictive computational model capable of recapitulating both encapsulation strategies is developed. The model is employed to comparatively study the oxygen concentration at different levels of vascularization, transplanted cell density, and spatial distribution, as well as with codelivered adjuvant cells. The model is then validated to be predictive of experimental results of oxygen pressure and local and systemic drug biodistribution in a direct vascularization device with local immunosuppressant delivery. The model highlights that dense vascularization can minimize cell hypoxia while allowing for high cell loading density. In contrast, lower levels of vascularization allow for better drug localization reducing systemic dissemination. Overall, it is shown that this model can serve as a valuable tool for the development and optimization of platform technologies for cell encapsulation.

Development of immune cell delivery system using biodegradable injectable polymers for cancer immunotherapy

Immune cell delivery using injectable hydrogel attracts much attention for improving its therapeutic effect. Specifically, dendritic cells (DCs) are the trigger cells for immune responses, and DC vaccines are studied for improving cancer immunotherapy. Hydrogel-assisted cell delivery is expected to enhance the viability of the implanted cells. We recently reported temperature-responsive biodegradable injectable polymer (IP) formulation utilizing poly(ε-caprolactone-co-glycolide)-b-poly(ethylene glycol)(PEG)-b-poly(ε-caprolactone-co-glycolide) (tri-PCG). Tri-PCG-based IP was reported to exhibit immediate sol-to-gel transition in response to temperature increase, in vivo biodegradability, and excellent biocompatibility. In this study, tri-PCG-based IP was applied to DC delivery. IP encapsulated live DCs, and the DCs incorporated ovalbumin (OVA) as a model antigen and CpG-DNA (oligo DNA with adjuvant effect) in IP hydrogel. Results suggested that DCs encapsulated in IP hydrogel internalized OVA and CpG-DNA and DCs were maturated to present antigens to T cells. Moreover, subcutaneously injected tri-PCG-based IP prolonged the retention period of cell accumulation at injected sites. Tri-PCG IP hydrogel could release matured DCs as the degradation of the hydrogel progressed. Tri-PCG IP formulation improved treatment efficacy of OVA transfected mouse lymphoma (E.G7-OVA) tumor. Hence, tri-PCG IP is a promising platform for immune cell delivery.

A click chemistry-mediated all-peptide cell printing hydrogel platform for diabetic wound healing

High glucose-induced vascular endothelial injury is a major pathological factor involved in non-healing diabetic wounds. To interrupt this pathological process, we design an all-peptide printable hydrogel platform based on highly efficient and precise one-step click chemistry of thiolated γ-polyglutamic acid, glycidyl methacrylate-conjugated γ-polyglutamic acid, and thiolated arginine-glycine-aspartate sequences. Vascular endothelial growth factor 165-overexpressed human umbilical vein endothelial cells are printed using this platform, hence fabricating a living material with high cell viability and precise cell spatial distribution control. This cell-laden hydrogel platform accelerates the diabetic wound healing of rats based on the unabated vascular endothelial growth factor 165 release, which promotes angiogenesis and alleviates damages on vascular endothelial mitochondria, thereby reducing tissue hypoxia, downregulating inflammation, and facilitating extracellular matrix remodeling. Together, this study offers a promising strategy for fabricating tissue-friendly, high-efficient, and accurate 3D printed all-peptide hydrogel platform for cell delivery and self-renewable growth factor therapy.

A 3D-Printed Dual Driving Forces Scaffold with Self-Promoted Cell Absorption for Spinal Cord Injury Repair.

Stem cells play critical roles in cell therapies and tissue engineering for nerve repair. However, achieving effective delivery of high cell density remains a challenge. Here, a novel cell delivery platform termed the hyper expansion scaffold (HES) is developed to enable high cell loading. HES facilitated self-promoted and efficient cell absorption via a dual driving force model. In vitro tests revealed that the HES rapidly expanded 80-fold in size upon absorbing 2.6 million human amniotic epithelial stem cells (hAESCs) within 2min, representing over a 400% increase in loading capacity versus controls. This enhanced uptake benefited from macroscopic swelling forces as well as microscale capillary action. In spinal cord injury (SCI) rats, HES-hAESCs promoted functional recovery and axonal projection by reducing neuroinflammation and improving the neurotrophic microenvironment surrounding the lesions. In summary, the dual driving forces model provides a new rationale for engineering hydrogel scaffolds to facilitate self-promoted cell absorption. The HES platform demonstrates great potential as a powerful and efficient vehicle for delivering high densities of hAESCs to promote clinical treatment and repair of SCI.

Improved Neural Differentiation of Human-induced Pluripotent Stem Cell [hiPSCs] on a Novel Polyurethane-based Scaffold Containing Iron Oxide Nanoparticles [Fe2O3 NPs

Repair of the nervous system in humans has always been complicated and faced difficulties. Cell transplantation approaches using biocompatible scaffolds might be an attractive therapeutic strategy for neuronal regeneration. We designed a cell delivery platform based on polyurethane [PU] and modified it with iron oxide nanoparticles [Fe2O3 NPs] for neural induction of human-induced pluripotent stem cells [hiPSC]. Forskolin, IBMX, and different ratios of FBS were employed to induce neurogenesis of hiPSCs. Neural differentiations were assessed at the level of genes and proteins. As was shown by MTT colorimetric assay, the proliferation and viability of SNL 76/7 on PU/ Fe2O3 were superior in comparison with pure PU and Fe2O3. hiPSCs cultured with PU/Fe2O3 exhibited an elevated expression of β3-tubulin, MAP2, NSE, OLIG2, as compared to controls. Furthermore, Acridine Orange staining assured the survival and viability of hiPSCs after 14 days of differentiation. All in all, our findings pointed out the biocompatibility and positive regulatory effect of PU/Fe2O3 on neural markers. We believe this scaffold could be a potential candidate for future nerve differentiation applications.

Preparation of Cell-Loaded Microbeads as Stable and Injectable Delivery Platforms for Tissue Engineering

Cell transplants in therapeutic studies do not preserve their long-term function inside the donor body. In mesenchymal stem cell (MSC) transplants, transplanted cells disperse through the body and are prone to degradation by immune cells after the transplant process. Various strategies, such as usage of the immunosuppressive drugs to eliminate allograft rejection, are designed to increase the efficiency of cell therapy. Another strategy is the construction of biomimetic encapsulates using polymeric materials, which isolate stem cells and protect them from environmental effects. In this study, fibroblasts (L929) and MSCs were investigated for their improved viability and functionality once encapsulated inside the alginate microbeads under in vitro conditions for up to 12 days of incubation. Thus, uniform and injectable (<200 µm) cell-loaded microbeads were constructed by the electrostatically assisted spraying technique. Results showed that both L929 and MSCs cells continue their metabolic activity inside the microbeads during the incubation periods. Glucose consumption and lactic acid production levels of both cell lines were consistently observed. The released cell number on day 12 was found to be increased compared to day 0. Protein expression levels of both groups increased every day with the expected doubling rate. Hence, this strategy with a simple yet clever design to encapsulate either MSCs or L929 cells might outstand as a potential cell delivery platform for cell therapy-based tissue engineering.

Microstructured click hydrogels for cell contact guidance in 3D.

The topography of the extracellular matrix (ECM) is a major biophysical regulator of cell behavior. While this has inspired the design of cell-instructive biomaterials, the ability to present topographic cues to cells in a true 3D setting remains challenging, particularly in ECM-like hydrogels made from a single polymer. Herein, we report the design of microstructured alginate hydrogels for injectable cell delivery and show their ability to orchestrate morphogenesis via cellular contact guidance in 3D. Alginate was grafted with hydrophobic cyclooctyne groups (ALG-K), yielding amphiphilic derivatives with self-associative potential and ionic crosslinking ability. This allowed the formation of microstructured ALG-KH hydrogels, triggered by the spontaneous segregation between hydrophobic/hydrophilic regions of the polymer that generated 3D networks with stiffer microdomains within a softer lattice. The azide-reactivity of cyclooctynes also allowed ALG-K functionalization with bioactive peptides via cytocompatible strain-promoted azide-alkyne cycloaddition (SPAAC). Hydrogel-embedded mesenchymal stem cells (MSCs) were able to integrate spatial information and to mechano-sense the 3D topography, which regulated cell shape and stress fiber organization. MSCs clusters initially formed on microstructured regions could then act as seeds for neo-tissue formation, inducing cells to produce their own ECM and self-organize into multicellular structures throughout the hydrogel. By combining 3D topography, click functionalization, and injectability, using a single polymer, ALG-K hydrogels provide a unique cell delivery platform for tissue regeneration.

Waffle-inspired hydrogel-based macrodevice for spatially controlled distribution of encapsulated therapeutic microtissues and pro-angiogenic endothelial cells.

Macro-encapsulation systems for delivery of cellular therapeutics in diabetes treatment offer major advantages such as device retrievability and high cell packing density. However, microtissue aggregation and absence of vasculature have been implicated in the inadequate transfer of nutrients and oxygen to the transplanted cellular grafts. Herein, we develop a hydrogel-based macrodevice to encapsulate therapeutic microtissues positioned in homogeneous spatial distribution to mitigate their aggregation while concurrently supporting an organized intra-device network of vascular-inductive cells. Termed Waffle-inspired Interlocking Macro-encapsulation (WIM) device, this platform comprises two modules with complementary topography features that fit together in a lock-and-key configuration. The waffle-inspired grid-like micropattern of the "lock" component effectively entraps insulin-secreting microtissues in controlled locations while the interlocking design places them in a co-planar spatial arrangement with close proximity to vascular-inductive cells. The WIM device co-laden with INS-1E microtissues and human umbilical vascular endothelial cells (HUVECs) maintains desirable cellular viability in vitro with theencapsulated microtissues retaining their glucose-responsive insulin secretion while embedded HUVECs express pro-angiogenic markers. Furthermore, a subcutaneously implanted alginate-coated WIM device encapsulating primary rat islets achieves blood glucose control for 2 weeks in chemically induced diabetic mice. Overall, this macrodevice design lays foundation for a cell delivery platform, which has the potential to facilitate nutrients and oxygen transport to therapeutic grafts and thereby might lead to improved disease management outcome.

Zwitterionic Polysaccharide-Based Hydrogel Dressing as a Stem Cell Carrier to Accelerate Burn Wound Healing.

Stem cell therapy integrated with hydrogels has shown promising potential in wound healing. However, the existing hydrogels usually cannot reach the desired therapeutic efficacy for burn wounds due to the inadaptability to wound shape and weak anti-infection ability. Moreover, it is difficult to improve the environment for the survival and function of stem cells under complicated wound microenvironments. In this study, an injectable and self-healing hydrogel (DSC), comprising sulfobetaine-derived dextran and carboxymethyl chitosan, is fabricated through a Schiff-base reaction. Meanwhile, the DSC hydrogel shows high nonfouling properties, including resistance to bacteria and nonspecific proteins; moreover, the prepared hydrogel can provide a biomimetic microenvironment for cell proliferation whilst maintaining the stemness of adipose-derived stem cells (ADSCs) regardless of complex microenvironments. In burnt murine animal models, the ADSCs-laden hydrogel can significantly accelerate wound healing rate and scarless skin tissue regeneration through multiple pathways. Specifically, the ADSCs-laden DSC hydrogel can avoid immune system recognition and activation and thus reduce the inflammatory response. Moreover, the ADSCs-laden DSC hydrogel can promote collagen deposition, angiogenesis, and enhance macrophage M2 polarization in the wound area. In summary, sulfobetaine-derived polysaccharide hydrogel can serve as a versatile platform for stem cell delivery to promote burn wound healing.

Angiogenic stem cell delivery platform to augment post-infarction neovasculature and reverse ventricular remodeling

Many cell-based therapies are challenged by the poor localization of introduced cells and the use of biomaterial scaffolds with questionable biocompatibility or bio-functionality. Endothelial progenitor cells (EPCs), a popular cell type used in cell-based therapies due to their robust angiogenic potential, are limited in their therapeutic capacity to develop into mature vasculature. Here, we demonstrate a joint delivery of human-derived endothelial progenitor cells (EPC) and smooth muscle cells (SMC) as a scaffold-free, bi-level cell sheet platform to improve ventricular remodeling and function in an athymic rat model of myocardial infarction. The transplanted bi-level cell sheet on the ischemic heart provides a biomimetic microenvironment and improved cell–cell communication, enhancing cell engraftment and angiogenesis, thereby improving ventricular remodeling. Notably, the increased density of vessel-like structures and upregulation of biological adhesion and vasculature developmental genes, such as Cxcl12 and Notch3, particularly in the ischemic border zone myocardium, were observed following cell sheet transplantation. We provide compelling evidence that this SMC-EPC bi-level cell sheet construct can be a promising therapy to repair ischemic cardiomyopathy.

Mesenchymal Stem Cell Sheet Centrifuge-Assisted Layering Augments Pro-Regenerative Cytokine Production.

A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment stimulates mesenchymal stem cell (MSC) paracrine factor production. In this capacity, cell sheets not only function as 3D cell delivery platforms, but also prime MSC therapeutic paracrine capacity. This study introduces a “cell sheet multilayering by centrifugation” strategy to non-invasively augment MSC paracrine factor production. Cell sheets fabricated by temperature-mediated harvest were first centrifuged as single layers using optimized conditions of rotational speed and time. Centrifugation enhanced cell physical and biochemical interactions related to intercellular communication and matrix interactions within the single cell sheet, upregulating MSC gene expression of connexin 43, integrin β1, and laminin α5. Single cell sheet centrifugation triggered MSC functional enhancement, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10). Subsequent cell sheet stacking, and centrifugation generated cohesive, bilayer MSC sheets within 2 h, which could not be accomplished within 24 h by conventional layering methods. Conventional layering led to H1F-1α upregulation and increased cell death, indicating a hypoxic thickness limitation to this approach. Comparing centrifuged single and bilayer cell sheets revealed that layering increased VEGF production 10-fold, attributed to intercellular interactions at the layered sheet interface. The “MSC sheet multilayering by centrifugation” strategy described herein generates a 3D MSC-delivery platform with boosted therapeutic factor production capacity.

Biohybrid Response Microparticles Decorated with Trained-MSCs for Acute Liver Failure Recovery.

Microcarrier-based mesenchymal stem cells (MSCs) delivery have attracted increasing attention in acute liver failure (ALF) therapy, while there is still room for improvement in terms of improving cell loading efficiency, enhancing anti-inflammatory features, and controlling cell release. Here, novel lipopolysaccharide (LPS)-composited magnetic-thermal responsive inverse opal particles (MIOPs) are presented for the delivery of MSCs. The MIOPs are composed of a chitosan inverse opal skeleton filled with a hydrogel containing LPS, poly(N-isopropylacrylamide), and Fe3 O4 nanoparticles. Benefitting from the biocompatible chitosan component and the huge specific surface area, the resultant MIOPs can capture MSCs in a nondestructive way. Furthermore, LPS can be released from the MIOPs under the stimulation of an alternating magnetic field, by which the MSCs are activated to gain the feature of "trained immunity." Moreover, this process can be monitored in real-time by the structural color change of the MIOPs. With that, the MSCs-laden MIOPs are employed in rats with ALF, and they exhibit obvious anti-inflammatory and therapeutic efficacy superior to untrained MSCs. These performances make the MIOPs a distinctive cell delivery platform for clinical tissue recovery applications.

α/β-Peptides as Nanomolar Triggers of Lipid Raft-Mediated Endocytosis through GM1 Ganglioside Recognition

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/β-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

Acellular Myocardial Scaffolds and Slices Fabrication, and Method for Applying Mechanical and Electrical Simulation to Tissue Construct.

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit to myocardial infarction (MI) treatment. Here, we described an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae, myocardial slices as a biomimetic cell culture and delivery platform, and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulations for facilitating cardiac construct development.

Current approaches to management of vocal fold scar.

Vocal fold (VF) fibrosis remains an insoluble problem in most cases, with a severe impact on vocal quality and effort. This review examines current investigations and research strands that explore the understanding of VF wound healing and applied treatments for the management of VF scar. Recent work focused on VF fibrosis has examined wound healing in the glottis, fibrosis-modifying medication, and tissue engineering approaches that span cytokine and growth factor therapy, scaffold and cell delivery platforms, seeded scaffolds, conditioned media and stem cell therapy. Many show promise and may deliver improvements in the wound bed favouring less fibrogenic healing patterns, ultimately with the goal of preserving or restoring VF vibration. Further collaborative research is required that examines combined approaches, long term outcomes, better three-dimensional modelling of cell-cell interactions and delivery modalities for molecular therapies. VF fibrosis research continues to expand and explore a variety of mechanistic pathways in order to understand VF healing and identify novel and complementary targets for manipulation. Many different approaches show promise and may also offer synergistic benefits. Research continues to strive for healing that more closely resembles true VF architecture and function.