Fibrin Gel Research Articles

Real-time spatiotemporal characterization of mechanics and sonoporation of acoustic droplet vaporization in acoustically responsive scaffolds.

Phase-shift droplets provide a flexible and dynamic platform for therapeutic and diagnostic applications of ultrasound. The spatiotemporal response of phase-shift droplets to focused ultrasound, via the mechanism termed acoustic droplet vaporization (ADV), can generate a range of bioeffects. Although ADV has been used widely in theranostic applications, ADV-induced bioeffects are understudied. Here, we integrated ultra-high-speed microscopy, confocal microscopy, and focused ultrasound for real-time visualization of ADV-induced mechanics and sonoporation in fibrin-based, tissue-mimicking hydrogels. Three monodispersed phase-shift droplets-containing perfluoropentane (PFP), perfluorohexane (PFH), or perfluorooctane (PFO)-with an average radius of ∼6 μm were studied. Fibroblasts and tracer particles, co-encapsulated within the hydrogel, were used to quantify sonoporation and mechanics resulting from ADV, respectively. The maximum radial expansion, expansion velocity, induced strain, and displacement of tracer particles were significantly higher in fibrin gels containing PFP droplets compared to PFH or PFO. Additionally, cell membrane permeabilization significantly depended on the distance between the droplet and cell (d), decreasing rapidly with increasing d. Significant membrane permeabilization occurred when d was smaller than the maximum radius of expansion. Both ultra-high-speed and confocal images indicate a hyper-local region of influence by an ADV bubble, which correlated inversely with the bulk boiling point of the phase-shift droplets. The findings provide insight into developing optimal approaches for therapeutic applications of ADV.

A tissue-engineered model of the atherosclerotic plaque cap: Toward understanding the role of microcalcifications in plaque rupture.

Rupture of the cap of an atherosclerotic plaque can lead to thrombotic cardiovascular events. It has been suggested, through computational models, that the presence of microcalcifications in the atherosclerotic cap can increase the risk of cap rupture. However, the experimental confirmation of this hypothesis is still lacking. In this study, we have developed a novel tissue-engineered model to mimic the atherosclerotic fibrous cap with microcalcifications and assess the impact of microcalcifications on cap mechanics. First, human carotid plaque caps were analyzed to determine the distribution, size, and density of microcalcifications in real cap tissue. Hydroxyapatite particles with features similar to real cap microcalcifications were used as microcalcification mimics. Injected clusters of hydroxyapatite particles were embedded in a fibrin gel seeded with human myofibroblasts which deposited a native-like collagenous matrix around the particles, during the 21-day culture period. Second harmonic multiphoton microscopy imaging revealed higher local collagen fiber dispersion in regions of hydroxyapatite clusters. Tissue-engineered caps with hydroxyapatite particles demonstrated lower stiffness and ultimate tensile stress than the control group samples under uniaxial tensile loading, suggesting increased rupture risk in atherosclerotic plaques with microcalcifications. This model supports previous computational findings regarding a detrimental role for microcalcifications in cap rupture risk and can further be deployed to elucidate tissue mechanics in pathologies with calcifying soft tissues.

NAMPT-Improved Mitochondrial Function Alleviates Degenerative Rotator Cuff Tendinopathy in Aged Mice.

Age-related rotator cuff tendinopathy (RCT) is associated with increased rotator cuff tear and postoperative retear rates. This study aimed to determine whether nicotinamide phosphoribosyltransferase (NAMPT) can alleviate degenerative RCT and prevent postoperative retears by reversing mitochondrial dysfunction in aged mice. We assigned 32 young (4 months) and 64 aged (19 to 20 months) male wild-type C57BL/6 mice to young, aged, and aged NAMPT-treated (ANAMPT) groups (n = 32 each). Mice in the ANAMPT group underwent subacromial injection with NAMPT-loaded fibrin gel, whereas the other 2 groups were injected with fibrin gel alone. Histological staining and each of the biomechanical and mitochondrial function tests were performed using 8 samples each. Histological staining in the aged group revealed decreased cellularity, disrupted fiber architecture, and reduced type-I collagen content inside tendon tissues proximal to the enthesis, demonstrating the spontaneous development of age-related degenerative RCT. Compared with the young group, the maximum tendon-to-bone failure load (4.22 ± 0.81 versus 5.52 ± 0.81 N, p = 0.0106) and maximum suture cut-through force (0.83 ± 0.08 versus 1.07 ± 0.10 N, p = 0.0006) of degenerated tendon tissues in the aged group were significantly lower. Significantly reduced nicotinamide adenine dinucleotide (NAD + ) levels, adenosine triphosphate (ATP) production, and citrate synthase activity indicated that mitochondrial dysfunction was closely related to the development of the degenerative RCT. Furthermore, NAMPT-improved mitochondrial function alleviated age-induced degenerative histological changes and increased the maximum failure load (5.32 ± 0.68 N, p = 0.0375) and maximum suture cut-through force (0.99 ± 0.13 N, p = 0.0285). Spontaneously developed degenerative RCT in aged mice mimicked the clinical situation in elderly patients. NAMPT-improved mitochondrial function could alleviate age-induced degenerative RCT and prevent postoperative suture cut-through of tendons with degenerative RCT. This study confirmed the spontaneous development of degenerative RCT in aged mice, which will facilitate future studies of this condition. The results also suggest that NAMPT offers a novel therapeutic approach for treating age-related degenerative RCT.

Hybrid AI models allow label-free identification and classification of pancreatic tumor repopulating cell population

Human pancreatic cancer cell lines harbor a small population of tumor repopulating cells (TRCs). Soft 3D fibrin gel allows efficient selection and growth of these tumorigenic TRCs. However, rapid and high-throughput identification and classification of pancreatic TRCs remain technically challenging. Here, we developed deep learning (DL) models paired with machine learning (ML) models to readily identify and classify 3D fibrin gel-selected TRCs into sub-types. Using four different human pancreatic cell lines, namely, MIA PaCa-2, PANC-1, CFPAC-1, and HPAF-II, we classified 3 main sub-types to be present within the TRC population. Our best model was an Inception-v3 convolutional neural network (CNN) used as a feature extractor paired with a Support Vector Machine (SVM) classifier with radial basis function (rbf) kernel which obtained a test accuracy of 90%. In addition, we compared this hybrid method of supervised classification with other methods of supervised classifications and showed that our working model outperforms others. With the help of unsupervised machine learning algorithms, we also validated that the pancreatic TRC subpopulation can be clustered into 3 sub-types. Collectively, our robust model can detect and readily classify tumorigenic TRC subpopulation label-free in a high-throughput fashion which can be very beneficial in clinical settings.

Binding of Pentagalloyl Glucose to Aortic Wall Proteins: Insights from Peptide Mapping and Simulated Docking Studies.

Pentagalloyl glucose (PGG) is currently being investigated as a non-surgical treatment for abdominal aortic aneurysms (AAAs); however, the molecular mechanisms of action of PGG on the AAA matrix components and the intra-luminal thrombus (ILT) still need to be better understood. To assess these interactions, we utilized peptide fingerprinting and molecular docking simulations to predict the binding of PGG to vascular proteins in normal and aneurysmal aorta, including matrix metalloproteinases (MMPs), cytokines, and fibrin. We performed PGG diffusion studies in pure fibrin gels and human ILT samples. PGG was predicted to bind with high affinity to most vascular proteins, the active sites of MMPs, and several cytokines known to be present in AAAs. Finally, despite potential binding to fibrin, PGG was shown to diffuse readily through thrombus at physiologic pressures. In conclusion, PGG can bind to all the normal and aneurysmal aorta protein components with high affinity, potentially protecting the tissue from degradation and exerting anti-inflammatory activities. Diffusion studies showed that thrombus presence in AAAs is not a barrier to endovascular treatment. Together, these results provide a deeper understanding of the clinical potential of PGG as a non-surgical treatment of AAAs.

Inference of long-range cell-cell force transmission from ECM remodeling fluctuations

Cells sense, manipulate and respond to their mechanical microenvironment in a plethora of physiological processes, yet the understanding of how cells transmit, receive and interpret environmental cues to communicate with distant cells is severely limited due to lack of tools to quantitatively infer the complex tangle of dynamic cell-cell interactions in complicated environments. We present a computational method to systematically infer and quantify long-range cell-cell force transmission through the extracellular matrix (cell-ECM-cell communication) by correlating ECM remodeling fluctuations in between communicating cells and demonstrating that these fluctuations contain sufficient information to define unique signatures that robustly distinguish between different pairs of communicating cells. We demonstrate our method with finite element simulations and live 3D imaging of fibroblasts and cancer cells embedded in fibrin gels. While previous studies relied on the formation of a visible fibrous ‘band’ extending between cells to inform on mechanical communication, our method detected mechanical propagation even in cases where visible bands never formed. We revealed that while contractility is required, band formation is not necessary, for cell-ECM-cell communication, and that mechanical signals propagate from one cell to another even upon massive reduction in their contractility. Our method sets the stage to measure the fundamental aspects of intercellular long-range mechanical communication in physiological contexts and may provide a new functional readout for high content 3D image-based screening. The ability to infer cell-ECM-cell communication using standard confocal microscopy holds the promise for wide use and democratizing the method.

Control of blood capillary networks and holes in blood-brain barrier models by regulating elastic modulus of scaffolds.

The blood-brain barrier (BBB) is a type of capillary network characterized by a highly selective barrier, which restricts the transport of substances between the blood and nervous system. Numerous in vitro models of the BBB have been developed for drug testing, but a BBB model with controllable capillary structures remains a major challenge. In this study, we report for the first time a unique method of controlling the blood capillary networks and characteristic holes formation in a BBB model by varying the elastic modulus of a three-dimensional scaffold. The characteristic hole structures are formed by the migration of endothelial cells from the model surface to the interior, which have functions of connecting the model interior to the external environment. The hole depth increased, as the elastic modulus of the fibrin gel scaffold increased, and the internal capillary network length increased with decreasing elastic modulus. Besides, internal astrocytes and pericytes were also found to be important for inducing hole formation from the model surface. Furthermore, RNA sequencing indicated up-regulated genes related to matrix metalloproteinases and angiogenesis, suggesting a relationship between enzymatic degradation of the scaffolds and hole formation. The findings of this study introduce a new method of fabricating complex BBB models for drug assessment.

A Bioglass-Poly(lactic-co-glycolic Acid) Scaffold@Fibrin Hydrogel Construct to Support Endochondral Bone Formation.

Bone tissue engineering using stem cells to build bone directly on a scaffold matrix often fails due to lack of oxygen at the injury site. This may be avoided by following the endochondral ossification route; herein, a cartilage template is promoted first, which can survive hypoxic environments, followed by its hypertrophy and ossification. However, hypertrophy is so far only achieved using biological factors. This work introduces a Bioglass-Poly(lactic-co-glycolic acid@fibrin (Bg-PLGA@fibrin) construct where a fibrin hydrogel infiltrates and encapsulates a porous Bg-PLGA. The hypothesis is that mesenchymal stem cells (MSCs) loaded in the fibrin gel and induced into chondrogenesis degrade the gel and become hypertrophic upon reaching the stiffer, bioactive Bg-PLGA core, without external induction factors. Results show that Bg-PLGA@fibrin induces hypertrophy, as well as matrix mineralization and osteogenesis; it also promotes a change in morphology of the MSCs at the gel/scaffold interface, possibly a sign of osteoblast-like differentiation of hypertrophic chondrocytes. Thus, the Bg-PLGA@fibrin construct can sequentially support the different phases of endochondral ossification purely based on material cues. This may facilitate clinical translation by decreasing in-vitro cell culture time pre-implantation and the complexity associated with the use of external induction factors.

Fibrin glue delivery system containing rhein ameliorates intervertebral disc degeneration by anti-inflammatory efficacy

PurposeTo construct an injectable, sustained-release fibrin gel containing rhein to solve the problem of low bioavailability of rhein, and observe its efficacy in the treatment of intervertebral disc degeneration.MethodsThe fibrin gel containing rhein was first synthesized in advance. Subsequently, the materials were characterized by various experimental methods. Secondly, the degenerative cell model was constructed by stimulating nucleus pulposus cells with lipopolysaccharide (LPS), and the corresponding intervention treatment was carried out to observe the effect in vitro. Finally, the rat tail intervertebral disc was acupunctured by needles to establish the intervertebral disc degeneration model, and the effect of the material was observed through intradiscal injection.ResultsThe fibrin glue containing rhein (rhein@FG) showed good injectability, sustained release and biocompatibility. Rhein@FG can improve the LPS-induced inflammatory microenvironment, regulate ECM metabolic disorders of nucleus pulposus cells and aggregation of the NLRP3 inflammasome in vitro, and inhibit cell pyroptosis. Furthermore, in vivo experiments, rhein@FG effectively prevented needle puncture-induced intervertebral disc degeneration in rats.ConclusionsRhein@FG has better efficacy than rhein or FG alone due to its slow release and mechanical properties, which can be used as a potential replacement therapy for intervertebral disc degeneration.Graphical

Composite Fibrin/Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs.

Biomaterials may enhance neural repair after spinal cord injury (SCI) and testing their functionality in large animals is essential to achieve successful clinical translation. This work developed a porcine contusion/compression SCI model to investigate the consequences of myelotomy and implantation of fibrin gel containing biofunctionalized carbon microfibers (MFs). Fourteen pigs were distributed in SCI, SCI/myelotomy, and SCI/myelotomy/implant groups. An automated device was used for SCI. A dorsal myelotomy was performed on the lesion site at 1 day post-injury for removing cloths and devitalized tissue. Bundles of MFs coated with a conducting polymer and cell adhesion molecules were embedded in fibrin gel and used to bridge the spinal cord cavity. Reproducible lesions of about 1 cm in length were obtained. Myelotomy and lesion debridement caused no further neural damage compared to SCI alone but had little positive effect on neural regrowth. The MFs/fibrin gel implant facilitated axonal sprouting, elongation, and alignment within the lesion. However, the implant also increased lesion volume and was ineffective in preventing fibrosis, thus precluding functional neural regeneration. Our results indicate that myelotomy and lesion debridement can be advantageously used for implanting MF-based scaffolds. However, the implants need refinement and pharmaceuticals will be necessary to limit scarring.

Acoustic droplet vaporization for on-demand modulation of microporosity in smart hydrogels.

Microporosity in hydrogels is critical for directing tissue formation and function. We have developed a fibrin-based smart hydrogel, termed an acoustically responsive scaffold (ARS), which responds to focused ultrasound in a spatiotemporally controlled, user-defined manner. ARSs are highly flexible platforms due to the inclusion of phase-shift droplets and their tunable response to ultrasound through a mechanism termed acoustic droplet vaporization (ADV). Here, we demonstrated that ADV enabled consistent generation of micropores in ARSs, throughout the entire thickness (∼5.5 mm), utilizing perfluorooctane phase-shift droplets. Size characteristics of the generated micropores were quantified in response to critical parameters including acoustic properties, droplet size, and shear elastic modulus of fibrin using confocal microscopy. The findings showed that the length of the generated micropores correlated directly with excitation frequency, peak rarefactional pressure, pulse duration, droplet size, and indirectly with the shear elastic modulus of the fibrin matrix. The ADV-generated micropores in ARSs were further compared with cavitation-mediated micropores in fibrin gels without droplets. Additionally, the Keller-Miksis equation was used to predict an upper bound for micropore formation in ARSs at varying driving frequencies and droplet sizes. Finally, our in vivo studies showed that host cell migration following ADV-induced micropore formation was frequency-dependent, with up to 2.6 times higher cell migration at lower frequencies. Overall, these findings demonstrate a new potential application of ADV in hydrogels. STATEMENT OF SIGNIFICANCE: Interconnected micropores within a hydrogel can facilitate many cell-mediated processes. Most techniques for generating micropores are typically not biocompatible or do not enable controlled, in situ micropore formation. We used an ultrasound-based technique, termed acoustic droplet vaporization, to generate microporosity in smart hydrogels termed acoustically responsive scaffolds (ARSs). ARSs contain a fibrin matrix doped with a phase-shift droplet. We demonstrate that unique acoustic properties of phase-shift droplets can be tailored to yield spatiotemporally controlled, on-demand micropore formation. Additionally, the size characteristics of the ultrasound-generated micropores can be modulated by tuning ultrasound parameters, droplet properties, and bulk elastic properties of fibrin. Finally, we demonstrate significant, frequency-dependent host cell migration in subcutaneously implanted ARSs in mice following ultrasound-induced micropore formation in situ.

Hydrogel Coating Optimization to Augment Engineered Soft Tissue Mechanics in Tissue-Engineered Blood Vessels.

Tissue engineering has the advantage of replicating soft tissue mechanics to better simulate and integrate into native soft tissue. However, soft tissue engineering has been fraught with issues of insufficient tissue strength to withstand physiological mechanical requirements. This factor is due to the lack of strength inherent in cell-only constructs and in the biomaterials used for soft tissue engineering and limited extracellular matrix (ECM) production possible in cell culture. To address this issue, we explored the use of an ECM-based hydrogel coating to serve as an adhesive tool, as demonstrated in vascular tissue engineering. The efficacy of cells to supplement mechanical strength in the coating was explored. Specifically, selected coatings were applied to an engineered artery tunica adventitia to accurately test their properties in a natural tissue support structure. Multiple iterations of three primary hydrogels with and without cells were tested: fibrin, collagen, and gelatin hydrogels with and without fibroblasts. The effectiveness of a natural crosslinker to further stabilize and strengthen the hydrogels was investigated, namely genipin extracted from the gardenia fruit. We found that gelatin crosslinked with genipin alone exhibited the highest tensile strength; however, fibrin gel supported cell viability the most. Overall, fibrin gel coating without genipin was deemed optimal for its balance in increasing mechanical strength while still supporting cell viability and was used in the final mechanical and hydrodynamic testing assessments. Engineered vessels coated in fibrin hydrogel with cells resulted in the highest tensile strength of all hydrogel-coated groups after 14 d in culture, demonstrating a tensile strength of 11.9 ± 2.91 kPa, compared to 5.67 ± 1.37 kPa for the next highest collagen hydrogel group. The effect of the fibrin hydrogel coating on burst pressure was tested on our strongest vessels composed of human aortic smooth muscle cells. A significant increase from our previously reported burst pressure of 51.3 ± 2.19 mmHg to 229 ± 23.8 mmHg was observed; however, more work is needed to render these vessels compliant with mechanical and biological criteria for blood vessel substitutes.

Construction of enzyme digested holes on hydrogel surface inspired by cell migration processes

The construction of in vitro capillary network models for drug testing and toxicity evaluation has become a major challenge in the field of tissue engineering. Previously, we discovered a novel phenomenon of hole formation by endothelial cell migration on the surface of fibrin gels. Interestingly, the hole characteristics, such as depth and number, were strongly influenced by the gel stiffness, but the details of hole formation are not to be clarified.In this study, we tried to understand the effect of hydrogel stiffness on the hole formation by dropping collagenase solution onto the surface of the hydrogels because the endothelial cell migration was made possible by the metalloproteinases' digestion. We found that smaller hole structures were formed on stiffer fibrin gels, but larger ones were formed on softer fibrin gels after the hydrogel digestion of the collagenase. This is consistent with our previous results in experiments on hole structures formed by endothelial cells. Furthermore, deep and small hole structures were successfully obtained by optimizing the volume of collagenase solution and incubation time. This unique approach inspired by endothelial cell hole formation may provide new methods of fabricating hydrogels with opening hole structures.

Three-dimensional cultures of gingival fibroblasts on fibrin-based scaffolds for gingival augmentation: A proof-of-concept study

ObjectiveGingival tissue regeneration is associated with several challenges. Tissue engineering regenerates the different components of the tissues, providing three major elements: living cells, appropriate scaffolds, and tissue-inducing substances. This study aimed to regenerate the gingival connective tissue in vitro, using human gingival fibroblasts cultured in three-dimensional fibrin gel scaffolds. DesignHuman gingival fibroblasts were seeded in a novel three-dimensional fibrin gel scaffold and maintained in two media types: platelet lysate media (control) and collagen-stimulating media (test). Cellular viability and proliferation were assessed, and the production of collagen and other extracellular matrix components in these constructs was investigated and compared. ResultsHuman gingival fibroblasts cultured in three-dimensional cultures were metabolically active and proliferated in both media. Furthermore, histologic sections, scanning electron microscopy, and quantitative polymerase chain reaction confirmed the production of higher levels of collagen and other extracellular matrix fibers in three-dimensional constructs cultured in collagen-stimulating media. ConclusionsCulturing human gingival fibroblasts in a novel three-dimensional fibrin gel scaffold containing collagen-stimulating media resulted in a tissue-equivalent construct that mimics human gingival connective tissue. The impact of these results should be considered for further investigations, which may help to develop a compatible scaffold for gingival soft tissue regeneration and treatment of mucogingival deformities.

Composite Hemostat Spray Seals Post-Surgical Blood Burst and Ameliorates Bacteria-Arised Inflammation for Expediting Wound Healing

Postoperative bleeding and infection are the main causes of death. Although a commercial zeolite-represented hemostatic agent has been widely used, zeolite-induced thermal damages to tissues are unresolved, and antibacterial and wound healing are desirably demanded but challenging. Herein, a composite zeolite-based hemostat spray consisting of Ag-doped β zeolite (Ag-βZ) and coupled fibrinogen/thrombin (F/T) has been developed to seal postsurgical blood burst. Systematic experiments reveal that the F/T can be switched into fibrin gel as the physical barrier and cover Ag-βZ, which imparts the composite hemostats with the most robust hemostasis ability associated with the lowest bleeding amount and the shortest hemostasis time. Concurrently, the physical barrier can shield Ag-βZ from touching water, reduce solvothermal release, and significantly minimize temperature, consequently resulting in no obvious thermal effect on the surrounding tissues. Importantly, the in situ doping of Ag can produce reactive oxygen species to kill bacteria and ameliorate bacteria-aroused inflammation, which can be also leveraged to promote cell expansion for expediting wound healing. The clinically approved components guarantee biosafety, paving a solid foundation for clinical translation.

A simple and effective scaffold for mouse hepatic stellate cell primary culture.

Scaffold-based culture is necessary for hepatic stellate cells (HSCs) because HSCs are promptly autoactivated under plastic conditions. Our research aims to investigate the potential and role of fibrin scaffold in reducing autoactivation, maintaining cell function, and extending the in vitro culture time of primary HSCs. HSCs were isolated from BALB/c mice and cultured on the surface of plastic, Matrigel, and fibrin gel. HSC's characteristics, including recovery, morphology, proliferation, lipid droplet (LD) storage, and activation were evaluated. Cell recovery was 86%, 80%, and 60% in fibrin, Matrigel, and plastic, respectively (P < 0.05). HSCs cultured on a plastic dish were autoactivated until day 7 with high proliferation, loss of cytoplasmic LD lipid droplets, and increased expression of activation markers, including alpha-smooth muscle actin (α-sma) and collagen type I. In contrast, these phenomena were reduced in Matrigel and fibrin-based cultures (P < 0.05). HSC culture in fibrin scaffold was associated with altered expression of cell adhesion molecules, including increased E-cadherin and inhibited N-cadherin. HSCs were more stellate-like in morphology in fibrin than in the Matrigel scaffold. Interestingly, fibrin-scaffold-embedded culture was able to maintain HSC quiescent state for up to 14 days in vitro. Fibrin gel could provide a potential scaffold for primary HSC culture while preserving cell function and extending primary HSC in vitro culture time.NEW & NOTEWORTHY Fibrin gel is appropriate for maintaining quiescence characteristics in primary culture of mouse hepatic stellate cells. Embedded culture of hepatic stellate cells in fibrin gel simulates in vivo cell morphology. Stiffness and adhesion molecules of fibrin gel play a crucial role in the hepatic stellate cell's primary culture.

Regenerative engineering of long bones using the small molecule forskolin

Bone grafting procedures have become increasingly common in the United States, with approximately 500,000 cases occurring each year at a societal cost exceeding $2.4 billion. Recombinant human bone morphogenetic proteins (rhBMPs) are therapeutic agents that have been widely used by orthopedic surgeons to stimulate bone tissue formation alone and when paired with biomaterials. However, significant limitations such as immunogenicity, high production cost, and ectopic bone growth from these therapies remain. Therefore, efforts have been made to discover and repurpose osteoinductive small-molecule therapeutics to promote bone regeneration. Previously, we have demonstrated that a single-dose treatment with the small-molecule forskolin for just 24 h induces osteogenic differentiation of rabbit bone marrow-derived stem cells invitro, while mitigating adverse side effects attributed with prolonged small-molecule treatment schemes. In this study, we engineered a composite fibrin-PLGA [poly(lactide-co-glycolide)]-sintered microsphere scaffold for the localized, short-term delivery of the osteoinductive small molecule, forskolin. Invitro characterization studies showed that forskolin released out of the fibrin gel within the first 24 h and retained its bioactivity toward osteogenic differentiation of bone marrow-derived stem cells. The forskolin-loaded fibrin-PLGA scaffold was also able to guide bone formation in a 3-mo rabbit radial critical-sized defect model comparable to recombinant human bone morphogenetic protein-2 (rhBMP-2) treatment, as demonstrated through histological and mechanical evaluation, with minimal systemic off-target side effects. Together, these results demonstrate the successful application of an innovative small-molecule treatment approach within long bone critical-sized defects.

Concentrated bone marrow aspirate and application for the treatment of osteochondral lesion and outcome

As osteochondral defects heal spontaneously or as a result of marrow stimulation, a bone marrow clot develops inside the cartilage defect. In the clot-filled defect, MSCs from the subchondral bone move in, develop into chondrocytes and osteoblasts, and eventually create a repair tissue. It is believed that applying a bone marrow aspirate (BMA) to marrow stimulation will improve cartilage repair since it may serve as a source of growth factors that promote both chondrogenesis and cartilage repair. Furthermore, the BMA clot offers a three-dimensional environment that may help chondrogenesis even more while safeguarding the subchondral bone from structural changes. In this treatment, bone marrow aspirate concentrated cells (BMAC), hyaluronic acid, and fibrin gel are applied after microfracture. The surgical procedure involved debridement of the lesion, microfracture, and application of concentrated BMAC with HA and fibrin gel.

Tissue-Engineered Core-Shell Silk-Fibroin/Poly-l-Lactic Acid Nerve Guidance Conduit Containing Encapsulated Exosomes of Human Endometrial Stem Cells Promotes Peripheral Nerve Regeneration.

Nerve guide conduits (NGCs) have been shown to be less efficient than nerve autografts in peripheral nerve regeneration. To address this issue, we developed for the first time a novel tissue-engineered nerve guide conduit structure encapsulated with human endometrial stem cell (EnSC) derived exosomes, which promoted nerve regeneration in rat sciatic nerve defects. In this study, we initially indicated the long-term efficacy and safety impacts of newly designed double layered SF/PLLA nerve guide conduits. Then the regeneration effects of SF/PLLA nerve guide conduits containing exosomes derived from human EnSCs were evaluated in rat sciatic nerve defects. The human EnSC derived exosomes were isolated from the supernatant of human EnSC cultures and characterized. Subsequently, the human EnSC derived exosomes were encapsulated in constructed NGCs by fibrin gel. For in vivo studies, entire 10 mm peripheral nerve defects were generated in rat sciatic nerves and restored with NGC encapsulated with human EnSC derived exosomes (Exo-NGC group), nerve guide conduits, and autografts. The efficiency of the NGCs encapsulated with human EnSCs derived exosomes in assisting peripheral nerve regeneration was investigated and compared with other groups. The in vivo results demonstrated that encapsulated human EnSC derived exosomes in NGC (Exo-NGC) significantly benefitted nerve regeneration based on motor function, sensory reaction, and electrophysiological results. Furthermore, immunohistochemistry with histopathology results showed the formation of regenerated nerve fibers, along with blood vessels that newly were developed, as a result of the exosome functions in the Exo-NGC group. These outcomes illustrated that the newly designed core-shell SF/PLLA nerve guide conduit encapsulated with human EnSC derived exosomes enhanced the regeneration process of axons and improved the functional recovery of rat sciatic nerve defects. So, encapsulated human EnSC-derived exosomes in a core-shell SF/PLLA nerve guide conduit are a potential therapeutic cell-free treatment for peripheral nerve defects.

Comparative analysis of the residues of granular support bath materials on printed structures in embedded extrusion printing

Embedded extrusion printing facilitates the fabrication of complex biological structures using soft hydrogels that are challenging to construct using conventional manufacturing methods. While this targeting strategy is appealing, the residues of support materials on the printed objects have been overlooked. Here, we quantitatively compare the bath residues on fibrin gel fibers printed in granular gel baths that are conjugated with fluorescent probes for visualization, including physically crosslinked gellan gum (GG) and gelatin (GEL) baths and chemically crosslinked polyvinyl alcohol baths. Notably, all support materials can be detected on a microscopic scale, even on structures without any visible residues. Quantitative results indicate that baths with smaller size or lower shear viscosity show more and deeper diffusion into the extruded inks, and the removal efficiency of support materials depends mainly on the dissolving property of the granular gel baths. The residual amount of chemically cross-linked support materials on fibrin gel fibers is 28–70 μg mm−2, which is tens of times higher than physically cross-linked GG (7.5 μg mm−2) and GEL (0.3 μg mm−2) baths. Meanwhile, cross-sectional images suggest that most gel particles are distributed around the fiber surface, but a small amount is in the fiber center. Such bath residues or the blank pores created by the removal of gel particles induce changes in product surface morphology, physicochemical and mechanical properties, impeding cell adhesion. This study will draw attention to the effects of residual support materials on printed structures and encourage the development of new strategies to diminish these residues or to take advantage of the residual support baths to improve product performances.