Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Characterization and Safety Profile of a New Combined Advanced Therapeutic Medical Product Platelet Lysate-Based Fibrin Hydrogel for Mesenchymal Stromal Cell Local Delivery in Regenerative Medicine.

Adipose-derived mesenchymal stromal cells (ASC) transplant to recover the optimal tissue structure/function relationship is a promising strategy to regenerate tissue lesions. Because filling local tissue defects by injection alone is often challenging, designing adequate cell carriers with suitable characteristics is critical for in situ ASC delivery. The aim of this study was to optimize the generation phase of a platelet-lysate-based fibrin hydrogel (PLFH) as a proper carrier for in situ ASC implantation and (1) to investigate in vitro PLFH biomechanical properties, cell viability, proliferation and migration sustainability, and (2) to comprehensively assess the local in vivo PLFH/ASC safety profile (local tolerance, ASC fate, biodistribution and toxicity). We first defined the experimental conditions to enhance physicochemical properties and microscopic features of PLFH as an adequate ASC vehicle. When ASC were mixed with PLFH, in vitro assays exhibited hydrogel supporting cell migration, viability and proliferation. In vivo local subcutaneous and subgingival PLFH/ASC administration in nude mice allowed us to generate biosafety data, including biodegradability, tolerance, ASC fate and engraftment, and the absence of biodistribution and toxicity to non-target tissues. Our data strongly suggest that this novel combined ATMP for in situ administration is safe with an efficient local ASC engraftment, supporting the further development for human clinical cell therapy.

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix.

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend.

Preparation and Characterization of Plasma-Derived Fibrin Hydrogels Modified by Alginate di-Aldehyde.

Fibrin hydrogels are one of the most popular scaffolds used in tissue engineering due to their excellent biological properties. Special attention should be paid to the use of human plasma-derived fibrin hydrogels as a 3D scaffold in the production of autologous skin grafts, skeletal muscle regeneration and bone tissue repair. However, mechanical weakness and rapid degradation, which causes plasma-derived fibrin matrices to shrink significantly, prompted us to improve their stability. In our study, plasma-derived fibrin was chemically bonded to oxidized alginate (alginate di-aldehyde, ADA) at 10%, 20%, 50% and 80% oxidation, by Schiff base formation, to produce natural hydrogels for tissue engineering applications. First, gelling time studies showed that the degree of ADA oxidation inhibits fibrin polymerization, which we associate with fiber increment and decreased fiber density; moreover, the storage modulus increased when increasing the final volume of CaCl2 (1% w/v) from 80 µL to 200 µL per milliliter of hydrogel. The contraction was similar in matrices with and without human primary fibroblasts (hFBs). In addition, proliferation studies with encapsulated hFBs showed an increment in cell viability in hydrogels with ADA at 10% oxidation at days 1 and 3 with 80 µL of CaCl2; by increasing this compound (CaCl2), the proliferation does not significantly increase until day 7. In the presence of 10% alginate oxidation, the proliferation results are similar to the control, in contrast to the sample with 20% oxidation whose proliferation decreases. Finally, the viability studies showed that the hFB morphology was maintained regardless of the degree of oxidation used; however, the quantity of CaCl2 influences the spread of the hFBs.

Effect of Fibrin Concentration on the In Vitro Production of Dermo-Epidermal Equivalents.

Human plasma-derived bilayered skin substitutes were successfully used by our group to produce human-based in vitro skin models for toxicity, cosmetic, and pharmaceutical testing. However, mechanical weakness, which causes the plasma-derived fibrin matrices to contract significantly, led us to attempt to improve their stability. In this work, we studied whether an increase in fibrin concentration from 1.2 to 2.4 mg/mL (which is the useful fibrinogen concentration range that can be obtained from plasma) improves the matrix and, hence, the performance of the in vitro skin cultures. The results show that this increase in fibrin concentration indeed affected the mechanical properties by doubling the elastic moduli and the maximum load. A structural analysis indicated a decreased porosity for the 2.4 mg/mL hydrogels, which can help explain this mechanical behavior. The contraction was clearly reduced for the 2.4 mg/mL matrices, which also allowed for the growth and proliferation of primary fibroblasts and keratinocytes, although at a somewhat reduced rate compared to the 1.2 mg/mL gels. Finally, both concentrations of fibrin gave rise to organotypic skin cultures with a fully differentiated epidermis, although their lifespans were longer (25-35%) in cultures with more concentrated matrices, which improves their usefulness. These systems will allow the generation of much better in vitro skin models for the testing of drugs, cosmetics and chemicals, or even to "personalized" skin for the diagnosis or determination of the most effective treatment possible.

Development of fibrin hydrogel-based in vitro bioassay system for assessment of skin permeability to and pro-inflammatory activity mediated by zinc ion released from nanoparticles.

Nanoparticles (NPs) are promising products in industry and medicine due to their unique physicochemical properties. In particular, zinc oxide (ZnO) NPs are extensively incorporated into sunscreens to protect the skin from exposure to ultraviolet radiation. However, there are several health concerns about skin penetration and the resultant toxicity. As methodologies for evaluating NP toxicity are under development, it is difficult to fully assess the toxicity of ZnO NPs toward humans. In this study, we developed a platform to simultaneously detect skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs. First, we generated a stable reporter cell line expressing green fluorescent protein (GFP) under the control of interleukin-8 (IL-8) promoter activity. The expression levels of GFP induced by zinc reflected the endogenous IL-8 expression levels and the pro-inflammatory responses. Next, we found that fibrin hydrogel can reproduce permeability to zinc ion of a human skin equivalent model and is therefore a promising material to assess skin permeability to zinc ion. Then, we constructed a fibrin hydrogel-based in vitro bioassay system for the simultaneous detection of skin permeability to and pro-inflammatory activity mediated by zinc ion released from NPs by using a stable reporter cell line and a fibrin hydrogel layer. This bioassay system is a promising in vitro permeation test due to its technical simplicity and good predictability. Overall, we believe that our bioassay system can be widely used in the cosmetics and pharmaceutical industries.

Towards Mimicking the Fetal Liver Niche: The Influence of Elasticity and Oxygen Tension on Hematopoietic Stem/Progenitor Cells Cultured in 3D Fibrin Hydrogels.

Hematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin-/cKit+ cells, HSPCs (Lin-/Sca+/cKit+ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.

Transplantation of miR-219 overexpressed human endometrial stem cells encapsulated in fibrin hydrogel in spinal cord injury.

Oligodendrocyte (OL) loss and demyelination occur after spinal cord injury (SCI). Stimulation of remyelination through transplantation of myelinating cells may be effective in improving function. For the repair strategy to be successful, the selection of a suitable cell and maintaining cell growth when cells are injected directly to the site of injury is important. In addition to selecting the type of cell, fibrin hydrogel was used as a suitable tissue engineering scaffold for this purpose. To test the relationship between myelination and functional improvement, the human endometrial stem cells (hEnSCs) were differentiated toward oligodendrocyte progenitor cells (OPCs) using overexpression of miR-219. Adult female Wistar rats were used to induce SCI by using a compression model and were randomly assigned to the following four experimental groups: SCI, Vehicle, hEnSC, and OPC. Ten days after injury, miR-219 overexpressed hEnSC-derived OPCs encapsulated in fibrin hydrogel, as an injectable scaffold, were injected to the injury site. In this study, with a focus on promoting functional recovery after SCI, the Basso-Beattie-Bresnahan test was performed to evaluate the recovery of motor function every week for 10 weeks and the histological assay was then performed. Results showed that the rate of motor function recovery was significantly higher in OPC group compared to SCI and vehicle groups but no marked differences were found between OPC and hEnSC groups, although, the rate of myelination in the OPC group was significantly higher than the other groups. These results demonstrated that remyelination was not the cause of recovery of motor function.

A PEGylated fibrin hydrogel-based antimicrobial wound dressing controls infection without impeding wound healing.

Combat injuries are associated with a high incidence of infection, and there is a continuing need for improved approaches to control infection and promote wound healing. Due to the possible local and systemic adverse effects of standard 1% cream formulation (Silvadene), we had previously developed a polyethylene glycol (PEGylated) fibrin hydrogel (FPEG)-based wound dressing for the controlled delivery of silver sulfadiazine (SSD) entrapped in chitosan microspheres (CSM). In this study, we have evaluated the antimicrobial and wound healing efficacy of SSD-CSM-FPEG using a full-thickness porcine wound infected with Pseudomonas aeruginosa. Infected wounds treated with a one-time application of the SSD-CSM-FPEG wound dressing demonstrated significantly reduced bacterial bioburden over time (99·99% of reduction by day 11; P < 0·05) compared with all the other treatment groups. The epithelial thickness and granulation of the wound bed was significantly better on day 7 (150·9 ± 13·12 µm), when compared with other treatment groups. Overall, our findings demonstrate that the SSD-CSM-FPEG wound dressing effectively controls P. aeruginosa infection and promotes wound healing by providing a favourable environment that induces neovascularisation. Collectively, sustained release of SSD using fibrin hydrogel exhibited enhanced benefits when compared with the currently available SSD treatment, and this may have significant implications in the bacterial reduction of infected wounds in military and civilian populations.

Blood Coagulation-Inspired Fibrin Hydrogel for Portable Detection of Thrombin Based on Personal Glucometer.

As one of the biomarkers of coagulation system-related diseases, the detection of thrombin is of practical importance. Thus, this study developed a portable biosensor based on a personal glucometer for rapid detection of thrombin activity. Fibrinogen was used for the detection of thrombin, and the assay principle was inspired by the blood coagulation process, where thrombin hydrolyzes fibrinogen to produce a fibrin hydrogel, and the amount of invertase encapsulated in the fibrin hydrogel fluctuates in accordance with the activity of thrombin in the sample solution. The quantitative assay is conducted by measuring the amount of unencapsulated invertase available to hydrolyze the substrate sucrose, and the signal readout is recorded using a personal glucometer. A linear detection range of 0-0.8 U/mL of thrombin with a limit of detection of 0.04 U/mL was obtained based on the personal glucometer sensing platform. The results of the selectivity and interference experiments showed that the developed personal glucometer sensing platform is highly selective and accurate for thrombin activity. Finally, the reliability of the portable glucometer method for rapid thrombin detection in serum samples was investigated by measuring the recovery rate, which ranged from 92.8% to 107.7%. In summary, the fibrin hydrogel sensing platform proposed in this study offers a portable and versatile means for detecting thrombin using a personal glucometer. This approach not only simplifies the detection process, but also eliminates the need for large instruments and skilled operators, and substantially reduces detection costs.

Cryopreserved nanostructured fibrin-agarose hydrogels are efficient and safe hemostatic agents.

Uncontrolled bleeding during surgery is associated with high mortality and prolonged hospital stay, necessitating the use of hemostatic agents. Fibrin sealant patches offer an efficient solution to achieve hemostasis and improve patient outcomes in liver resection surgery. We have previously demonstrated the efficacy of a nanostructured fibrin-agarose hydrogel (NFAH). However, for the widespread distribution and commercialization of the product, it is necessary to develop an optimal preservation method that allows for prolonged stability and facilitates storage and distribution. We investigated cryopreservation as a potential method for preserving NFAH using trehalose. Structural changes in cryopreserved NFAH (Cryo-NFAH) were investigated and comparative in vitro and in vivo efficacy and safety studies were performed with freshly prepared NFAH. We also examined the long-term safety of Cryo-NFAH versus TachoSil in a rat partial hepatectomy model, including time to hemostasis, intra-abdominal adhesion, hepatic hematoma, inflammatory factors, histopathological variables, temperature and body weight, hemocompatibility and cytotoxicity. Structural analyses demonstrated that Cryo-NFAH retained most of its macro- and microscopic properties after cryopreservation. Likewise, hemostatic efficacy assays showed no significant differences with fresh NFAH. Safety evaluations indicated that Cryo-NFAH had a similar overall profile to TachoSil up to 40 days post-surgery in rats. In addition, Cryo-NFAH demonstrated superior hemostatic efficacy compared with TachoSil while also demonstrating lower levels of erythrolysis and cytotoxicity than both TachoSil and other commercially available hemostatic agents. These results indicate that Cryo-NFAH is highly effective hemostatic patch with a favorable safety and tolerability profile, supporting its potential for clinical use.

Fibrin Hydrogel Layer-Anchored Pericardial Matrix Prevents Epicardial Adhesion in the Severe Heart Adhesion-Induced Miniature Pig Model.

Postoperative adhesion is a very common and serious complication that occurs frequently in cardiac surgery. The purpose of this study was to evaluate the efficacy of a fibrin hydrogel layer-anchored decellularized pericardial matrix in preventing pericardial adhesions in a miniature pig model with a myocardial injury. Fibrin hydrogel layer-anchored decellularized pericardial matrix was prepared by spraying a mixture of fibrinogen and thrombin on a fibrinogen-doped decellularized pericardium. Cardiac injury was generated by abrading and desiccating the epicardial surface of a miniature pig to induce severe postoperative adhesions. The adhesion between the epicardial surface and fibrin hydrogel layer-anchored decellularized pericardial matrix in three different regions (left outer, front, and right outer) was evaluated macroscopically one month after surgery. The fibrin hydrogel layer-anchored decellularized pericardial matrix showed significantly less adhesion than an autologous pericardium (0.2 ± 0.7 in DPM-FHG0.5 and 0.4 ± 0.8 in DPM-FHG1, p < 0.01) and expanded polytetrafluoroethylene (ePTFE) (1.6 ± 0.5, p < 0.05). The fibrin hydrogel concentration had no effect on preventing postoperative adhesion. A thinner fibrin hydrogel layer was observed on the decellularized pericardial matrix one month after surgery; however, the inside of the matrix was filled with fibrin hydrogel. Fibrin hydrogel layer-anchored decellularized pericardial matrix prevented postoperative epicardial adhesions in a miniature pig model. Our findings suggest that pericardial closure using a fibrin hydrogel layer-anchored decellularized pericardial matrix is a promising method for preventing adverse outcomes in reoperative surgeries.

Repair of Rat Calvarial Critical-Sized Defects Using Heparin-Conjugated Fibrin Hydrogel Containing BMP-2 and Adipose-Derived Pericytes.

The repair of critical-sized calvarial defects is a challenging problem for orthopedic surgery. One of the promising strategies of bone bioengineering to enhance the efficacy of large bone defect regeneration is the combined delivery of stem cells with osteoinductive factors within polymer carriers. The purpose of the research was to study the regenerative effects of heparin-conjugated fibrin (HCF) hydrogel containing bone morphogenetic protein 2 (BMP-2) and adipose-derived pericytes (ADPs) in a rat critical-sized calvarial defect model. In vitro analysis revealed that the HCF hydrogel was able to control the BMP-2 release and induce alkaline phosphatase (ALP) activity in neonatal rat osteoblasts. In addition, it was found that eluted BMP-2 significantly induced the osteogenic differentiation of ADPs. It was characterized by the increased ALP activity, osteocalcin expression and calcium deposits in ADPs. In vivo studies have shown that both HCF hydrogel with BMP-2 and HCF hydrogel with pericytes are able to significantly increase the regeneration of critical-sized calvarial defects in comparison with the control group. Nevertheless, the greatest regenerative effect was found after the co-delivery of ADPs and BMP-2 into a critical-sized calvarial defect. Thus, our findings suggest that the combined delivery of ADPs and BMP-2 in HCF hydrogel holds promise to be applied as an alternative biopolymer for the critical-sized bone defect restoration.

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.

Sleeping Beauty transposon system for GDNF overexpression of entrapped stem cells in fibrin hydrogel in a rat model of Parkinson's disease.

There is currently no causal treatment available for Parkinson's disease (PD). However, the use of glial cell line-derived neurotrophic factor (GDNF) to provide regenerative effects for neurons is promising. Such approaches require translational delivery systems that are functional in diseased tissue. To do so, we used a non-viral Sleeping Beauty (SB) transposon system to overexpress GDNF in adipose tissue-derived mesenchymal stromal cells (adMSCs). Entrapment of cells in fibrin hydrogel was used to boost potential neurorestorative effects. Functional GDNF-adMSCs were able to secrete 1066.8 ± 169.4 ng GDNF/120,000 cells in vitro. The GDNF-adMSCs were detectable for up to 1 month after transplantation in a mild 6-hydroxydopamine (6-OHDA) hemiparkinson male rat model. Entrapment of GDNF-adMSCs enabled GDNF secretion in surrounding tissue in a more concentrated manner, also tending to prolong GDNF secretion relatively. GDNF-adMSCs entrapped in hydrogel also led to positive immunomodulatory effects via an 83% reduction of regional IL-1ß levels compared to the non-entrapped GDNF-adMSC group after 1 month. Furthermore, GDNF-adMSC-treated groups showed higher recovery of tyrosine hydroxylase (TH)-expressing cells, indicating a neuroprotective function, although this was not strong enough to show significant improvement in motor performance. Our findings establish a promising GDNF treatment system in a PD model. Entrapment of GDNF-adMSCs mediated positive immunomodulatory effects. Although the durability of the hydrogel needs to be extended to unlock its full potential for motor improvements, the neuroprotective effects of GDNF were evident and safe. Further motor behavioral tests and other disease models are necessary to evaluate this treatment option adequately.

Structural alignment guides oriented migration and differentiation of endogenous neural stem cells for neurogenesis in brain injury treatment.

Radial glia (RG) cells that align in parallel in the embryonic brain are found to be able to guide the directed migration of neurons in response to brain injury. Therefore, biomaterials with aligned architectures are supposed to have positive effects on neural migration and neurogenic differentiation for brain injury repair that are rarely addressed, although they have been widely demonstrated in spinal cord and peripheral nerve system. Here, we present a highly biomimetic scaffold of aligned fibrin hydrogel (AFG) that mimics the oriented structure of RG fibers. Through a combination of histological, behavioral, imaging, and transcriptomic analyses, we demonstrated that transplanting the AFG scaffold into injured cortical brains promotes effective migration, differentiation, and maturation of endogenous neural stem cells, resulting in neurological functional recovery. Therefore, this study will light up a new perspective on applying an aligned scaffold to promote cortical regeneration after injury by inducing endogenous neurogenesis.

Endostatin in 3D Fibrin Hydrogel Scaffolds Promotes Chondrogenic Differentiation in Swine Neonatal Meniscal Cells.

The success of cell-based approaches for the treatment of cartilage or fibro-cartilaginous tissue defects requires an optimal cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. For this purpose, the aim of this study was to evaluate the use of endostatin (COL18A1), an anti-angiogenic factor, which is physiologically involved in cell differentiation during meniscus development. Swine neonatal meniscal cells not yet subjected to mechanical stimuli were extracted, cultured in fibrin hydrogel scaffolds, and treated at two different time points (T1 = 9 days and T2 = 21 days) with different concentrations of COL18A1 (10 ng/mL; 100 ng/mL; 200 ng/mL). At the end of the treatments, the scaffolds were examined through biochemical, molecular, and histochemical analyses. The results showed that the higher concentration of COL18A1 promotes a fibro-chondrogenic phenotype and improves cellularity index (DNA content, p < 0.001) and cell efficiency (GAGs/DNA ratio, p < 0.01) after 21 days. These data are supported by the molecular analysis of collagen type I (COL1A1, a marker of fibrous-like tissue, p < 0.001), collagen type II (COL2A1, a marker of cartilaginous-like tissue, p < 0.001) and SRY-Box Transcription Factor 9 (SOX9, an early marker of chondrogenicity, p < 0.001), as well as by histological analysis (Safranin-O staining), laying the foundations for future studies evaluating the involvement of 3D endostatin hydrogel scaffolds in the differentiation of avascular tissues.

White matter regeneration induced by aligned fibrin nanofiber hydrogel contributes to motor functional recovery in canine T12 spinal cord injury.

A hierarchically aligned fibrin hydrogel (AFG) that possesses soft stiffness and aligned nanofiber structure has been successfully proven to facilitate neuroregeneration in vitro and in vivo. However, its potential in promoting nerve regeneration in large animal models that is critical for clinical translation has not been sufficiently specified. Here, the effects of AFG on directing neuroregeneration in canine hemisected T12 spinal cord injuries were explored. Histologically obvious white matter regeneration consisting of a large area of consecutive, compact and aligned nerve fibers is induced by AFG, leading to a significant motor functional restoration. The canines with AFG implantation start to stand well with their defective legs from 3 to 4 weeks postoperatively and even effortlessly climb the steps from 7 to 8 weeks. Moreover, high-resolution multi-shot diffusion tensor imaging illustrates the spatiotemporal dynamics of nerve regeneration rapidly crossing the lesion within 4 weeks in the AFG group. Our findings indicate that AFG could be a potential therapeutic vehicle for spinal cord injury by inducing rapid white matter regeneration and restoring locomotion, pointing out its promising prospect in clinic practice.

Regeneration of Osteochondral Defects by Combined Delivery of Synovium-Derived Mesenchymal Stem Cells, TGF-ß1 and BMP-4 in Heparin-Conjugated Fibrin Hydrogel.

The regeneration of cartilage and osteochondral defects remains one of the most challenging clinical problems in orthopedic surgery. Currently, tissue-engineering techniques based on the delivery of appropriate growth factors and mesenchymal stem cells (MSCs) in hydrogel scaffolds are considered as the most promising therapeutic strategy for osteochondral defects regeneration. In this study, we fabricated a heparin-conjugated fibrin (HCF) hydrogel with synovium-derived mesenchymal stem cells (SDMSCs), transforming growth factor-ß1 (TGF-ß1) and bone morphogenetic protein-4 (BMP-4) to repair osteochondral defects in a rabbit model. An in vitro study showed that HCF hydrogel exhibited good biocompatibility, a slow degradation rate and sustained release of TGF-ß1 and BMP-4 over 4 weeks. Macroscopic and histological evaluations revealed that implantation of HCF hydrogel with SDMSCs, TGF-ß1 and BMP-4 significantly enhanced the regeneration of hyaline cartilage and the subchondral bone plate in osteochondral defects within 12 weeks compared to hydrogels with SDMSCs or growth factors alone. Thus, these data suggest that combined delivery of SDMSCs with TGF-ß1 and BMP-4 in HCF hydrogel may synergistically enhance the therapeutic efficacy of osteochondral defect repair of the knee joints.

Loss of sarcomeric proteins via upregulation of JAK/STAT signaling underlies interferon-γ-induced contractile deficit in engineered human myocardium.

The level of circulating interferon-γ (IFNγ) is elevated in various clinical conditions including autoimmune and inflammatory diseases, sepsis, acute coronary syndrome, and viral infections. As these conditions are associated with high risk of myocardial dysfunction, we investigated the effects of IFNγ on 3D fibrin-based engineered human cardiac tissues ("cardiobundles"). Cardiobundles were fabricated from human pluripotent stem cell-derived cardiomyocytes, exposed to 0-20 ng/ml of IFNγ on culture days 7-14, and assessed for changes in tissue structure, viability, contractile force and calcium transient generation, action potential propagation, cytokine secretion, and expression of select genes and proteins. We found that application of IFNγ induced a dose-dependent reduction in contractile force generation, deterioration of sarcomeric organization, and cardiomyocyte disarray, without significantly altering cell viability, action potential propagation, or calcium transient amplitude. At molecular level, the IFNγ-induced structural and functional deficits could be attributed to altered balance of pro- and anti-inflammatory cytokines, upregulation of JAK/STAT signaling pathway (JAK1, JAK2, and STAT1), and reduced expression of myosin heavy chain, myosin light chain-2v, and sarcomeric α-actinin. Application of clinically used JAK/STAT inhibitors, tofacitinib and baricitinib, fully prevented IFNγ-induced cardiomyopathy, confirming the critical roles of this signaling pathway in inflammatory cardiac disease. Taken together, our in vitro studies in engineered myocardial tissues reveal direct adverse effects of pro-inflammatory cytokine IFNγ on human cardiomyocytes and establish the foundation for a potential use of cardiobundle platform in modeling of inflammatory myocardial disease and therapy. STATEMENT OF SIGNIFICANCE: Various inflammatory and autoimmune diseases including rheumatoid arthritis, sepsis, lupus erythematosus, Chagas disease, and others, as well as viral infections including H1N1 influenza and COVID-19 show increased systemic levels of a pro-inflammatory cytokine interferon-γ (IFNγ) and are associated with high risk of heart disease. Here we explored for the first time if chronically elevated levels of IFNγ can negatively affect structure and function of engineered human heart tissues in vitro. Our studies revealed IFNγ-induced deterioration of myofibrillar organization and contractile force production in human cardiomyocytes, attributed to decreased expression of multiple sarcomeric proteins and upregulation of JAK/STAT signaling pathway. FDA-approved JAK inhibitors fully blocked the adverse effects of IFNγ, suggesting a potentially effective strategy against human inflammatory cardiomyopathy.