While acute wounds typically progress through the phases of wound healing, chronic wounds often stall in the inflammatory phase due to elevated levels of matrix metalloproteinases (MMPs) and proinflammatory cytokines. Dysregulated expression of MMPs can result in the breakdown of extracellular matrix (ECM) formed during the wound healing process, resulting in stalled wounds. Native collagen-based wound dressings offer a potential wound management option to sequester excess MMPs and support cellular interactions that allow wound progression through the natural healing process. Herein, we utilized commercially available ECM matrices, two derived from porcine small intestinal submucosa (PCMP, 2 layers; PCMP-XT, 5 layers) and one derived from propria submucosa (ovine forestomach matrix, OFM, 1 layer), to demonstrate the impact of processing methodologies (e.g., layering and crosslinking) on functional characteristics needed for the management of chronic wounds. Grafts were evaluated for structural composition using scanning electron microscopy and histology, ability to reduce MMPs using fluorometric assays, and durability in an degradation chronic wound model. Both intact (nondegraded) and partially degraded grafts were assessed for their ability to serve as a functional cell scaffold using primary human fibroblasts. Grafts differed in matrix substructure and composition. While all grafts demonstrated attenuation of MMP activity, PCMP and PCMP-XT showed larger reductions of MMP levels. OFM rapidly degraded in the degradation model (<3 hours), while PCMP and PCMP-XT were significantly more durable (>7 days). The ability of PCMP and PCMP-XT to serve as scaffolds for cellular attachment was not impacted by degradation . Three ECM grafts with varying structural and functional characteristics exhibited differential durability when degraded in a simulated chronic wound model. Those that withstood rapid degradation maintained their ability to function as a scaffold to support attachment and proliferation of fibroblasts, a cell type important for wound healing.

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed , which included cell migration, viability, and apoptosis. , a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts . Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. , increased bone volume per tissue volume (BV/TV) (=0.031), trabecular thickness (Tb.Th) (=0.020), and decreased separation (Tb.Sp) (=0.040) were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

Wound healing is an intricate process involving multiple cells and distinct phases, presenting challenges for comprehensive investigations. Currently available treatments for wounds have limited capacity to fully restore tissue and often require significant investments of time in the form of repetitive dressing changes and/or reapplications. This article presents a novel study that aims to enhance wound healing by developing biomaterial scaffolds using Medpor®, a porous polyethylene implant, as a model scaffold. The study incorporates electrospun poly(e-caprolactone) (PCL) fibers and a protein mixture (PM) containing collagen IV and laminin onto the Medpor® scaffolds. To evaluate the impact of these implants on wound healing, a unique splinted wound model in mice is employed. The wounds were evaluated for closure, inflammation, collagen deposition, angiogenesis, epithelialization, and proliferation. The results show that wounds treated with Medpor® + PCL + PM implants demonstrate accelerated closure rates, improved epithelialization, and enhanced angiogenesis compared to other implant groups. However, there were no significant differences observed in collagen deposition and inflammatory response among the implant groups. This study provides valuable insights into the potential benefits of incorporating PCL fibers and a PM onto scaffolds to enhance wound healing. Furthermore, the developed splinted wound model with integrated implants offers a promising platform for future studies on implant efficacy and the advancement of innovative wound healing strategies.

Peripheral neuropathy is painful and can cause a considerable decline in quality of life. Surgery and autograft are the current approaches and clinical standards for restoring function after nerve damage. However, they usually result in unacceptable clinical results, so we need modern peripheral nerve defect treatment approaches. Tissue engineering techniques have been developed as a promising approach, but there are some considerations for translational application. Clinical application of novel tissue engineering methods is related to combining the appropriate cell and scaffold type to introduce safe and efficient bioscaffolds. Efficient nerve regeneration occurs by mimicking the extracellular matrix and combining topographical, biochemical, mechanical, and conductive signs via different cells, biomolecules, and polymers. In brief, ideal engineered biomaterial scaffolds will have to cover all characteristics of nerve tissue, such as nerve number, myelin, and axon thickness. Nerve regeneration has a highly sensitive response to its surrounding microenvironment. For designing a suitable construct, matching the regenerative potential of the autograft as the golden standard is essential. This review article examines the newest advancements in peripheral nerve tissue engineering. Specifically, the discussion will focus on incorporating innovative cues, biological modification, biomaterials, techniques, and concepts in this area of research.

The decellularized human amniotic membrane (dHAM) emerges as a viable 3D scaffold for organ repair and replacement using a tissue engineering strategy. Glutaraldehyde (GTA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) can increase the biomechanical properties of dHAM. However, the crosslinking process is associated with biochemical changes and residual toxic materials, dampening the biocompatibility of the dHAM. From a histologic point of view, each side of the amniotic membrane is biologically different. While the dHAM basement membrane side is rich in growth factors, the stromal side of the dHAM contains more connective tissue matrix (e.g., collagen fibers) which supports its biomechanical properties. Biocompatibility and biomechanical properties are two important challenges in the field of materials science. In this study, for the first time, the stromal and basement membrane side are cross-linked with GTA and EDC, respectively, to optimize the biocompatibility of the treated dHAM while sparing the GTA-mediated biomechanical improvements.

[This retracts the article DOI: 10.1002/term.3054.].

Despite ongoing efforts, the regeneration of critical-sized bone defects remains a significant challenge for clinicians due to the absence of a standard clinically compliant bone tissue engineering protocol. These challenges are mostly attributed to the inadequacies of current methods, characterized by their high variability and the reliance on animal-derived components, such as fetal bovine serum (FBS) in cell culture. To address these shortcomings, our approach diverges from conventional practices by prioritizing consistency and reproducibility, and the complete elimination of animal derivatives throughout the entire process. We have developed a novel method that utilizes a peptide-functionalized photocrosslinkable methacrylated hyaluronic acid (MeHA-RGD) hydrogel as a cell sealant for loading human adipose-derived stem cells (hASCs) into a 3D porous polycaprolactone-tricalcium phosphate (PCL-TCP) scaffold. Additionally, we created a 3D-printed vacuum-assisted cell loading device to facilitate this process and ensure efficiency and consistency during cell loading. Our findings indicate that the MeHA-RGD hydrogel supports both stem cell viability and osteogenic differentiation, demonstrating outcomes comparable to those achieved with fibrin glue, a conventional cell sealant widely used in BTE from autologous or xenogeneic sources, even under serum- and xeno-free conditions. In the pursuit of clinical translation, it is vital that biomaterials exhibit low variability, are easily accessible, readily available, and completely free of animal derivatives. To our knowledge, this is the first study to employ a 3D-printed vacuum-assisted cell loading device system within a fully defined hybrid 3D system under complete serum- and xeno-free conditions. These findings unravel and encourage alternative approaches in addressing the existing challenges in BTE, thereby facilitating and accelerating clinical translation in the future.

Saethre-Chotzen syndrome (SCS) is one of the most prevalent craniosynostosis, caused by a loss-of-function mutation in the gene, with current treatment options relying on major invasive transcranial surgery. haploinsufficient osteogenic progenitor cells exhibit increased osteogenic differentiation potential due to an upregulation of the transmembrane tyrosine kinase receptor, , a TWIST-1 target gene known to promote bone formation. The present study assessed the efficacy of suppressing C-ROS-1 activity using a known chemical inhibitor to C-ROS-1, crizotinib, to halt premature coronal suture fusion in a preclinical mouse model of SCS. Crizotinib (1 M, 2 M, or 4 M) was administered locally over the calvaria of Twist-1 heterozygous mice prior to coronal suture fusion using either a nonresorbable collagen sponge (quick drug release) or a resorbable sodium carboxymethylcellulose microdisk (slow sustained release). Coronal suture fusion rates and bone parameters were determined by CT imaging and histomorphometric analysis of calvaria postcoronal suture fusion. Results demonstrated a dose-dependent increase in the efficacy of crizotinib to maintain coronal suture patency, with no adverse effects to brain, kidney, liver, and spleen tissue, or blood cell parameters. Moreover, crizotinib delivered on microdisks resulted in a greater efficacy at a lower concentration to reduce bone formation at the coronal suture sites compared to sponges. However, the bone inhibitory effects were found to be diminished by over time following cessation of treatment. Our findings lay the foundation for the development of a pharmacological nonsurgical, targeted approach to temporarily maintain open coronal sutures in SCS patients. This study could potentially be used to develop similar therapeutic strategies to treat different syndromic craniosynostosis conditions caused by known genetic mutations.

[This retracts the article DOI: 10.1002/term.3184.].

Regenerative medicine (RM) exploits stem cells to construct biological replacements and repair damaged tissues, offering an alternative to daunting organ transplantation. Even while RM has advanced quickly, building an entire organ remains beyond our capabilities. Experts are thus investigating the adoption of biologically generated composites that preserve the tissue's crucial physiological, morphological, and mechanical characteristics. Noncellular tissues like extracellular matrix offer cells a milieu similar to their physiological niche, becoming a promising substitute for synthetic composites. In this context, amnion, the membrane enclosing the fetus, is a great contender since it is widely obtainable and economical. Given its biochemical and anatomic characteristics, and the extensive supply of stem cells, growth factors, and matrix proteins, the amnion is considered a fantastic candidate to employ in RM. Decellularized amniotic membrane (DAM) has many uses as two- and three-dimensional scaffolds, anchoring for cell adhesion and expansion for tissue regeneration, and as carrier systems for cell and drug cargoes. The present research aims to assess the recent surge in DAM-RM research, potentially to get beyond the existing barriers impeding the RM's clinical translation landscape. The present paper draws a comprehensive picture of the experimental evidence and clinical trials regarding exploiting DAM in RM.

[This retracts the article DOI: 10.1002/term.3295.].

Ex vivo organ perfusion (EVOP) is used for whole organ preservation, and the main focus is to improve the outcome of donor organs for transplantation. Recently, EVOP has found application in disease modeling, drug development, and tissue regeneration. We discuss progress in EVOP research involving small animal organs using benchtop and incubator-based EVOP systems, highlighting innovative designs of EVOP systems, technical specifications of each system, and their versatile applications across a range of research fields.

Retinal degeneration is the leading cause of blindness worldwide. Subretinal implantation of human retinal progenitor cells (hRPCs) has shown great promise in models of retinal degeneration for restoration of vision but is limited by extremely low (< 2%) integration into the retina. Successful integration of implanted cells requires their migration from the site of implantation into the degenerating retina. Little is known about what cues promote RPC migration in the context of the postimplantation microenvironment, such as cues presented by a biomaterial carrier. We utilized a high-throughput assay to study the migration of hRPCs in three-dimensional hydrogel matrices of varying chemical composition and stiffness and, with exposure to different soluble factors, to identify cues important for hRPC migration and associated cell signaling events driving migration. Collagen type I, collagen type I methacrylate, and hyaluronic acid glycidyl methacrylate gels were developed with variable stiffness. The impact of key growth factors in neural development, regeneration, and cell migration such as epidermal growth factor (EGF), fibroblast growth factor (FGF), stromal cell-derived factor (SDF), and hepatocyte growth factor (HGF) was studied using hRPCs in 2 mg/mL collagen type I gels. Migration of the hRPCs varied significantly in gels of different composition and stiffness, with higher levels of mean migration distance after 48 h in nonphoto crosslinked collagen-based gels with higher concentrations of gel components and associated compressive moduli. In addition, the presence of SDF and HGF in collagen gels increased hRPC migration compared to media alone. Key signaling nodes correlating with hRPC migration were identified in Akt and MAPK signaltransduction pathways using bead-based multiplex ELISA and partial least-squares regression (PLSR) modeling. These results motivate the further exploration of material stiffness and co-delivery of soluble factors as important design parameters in cell delivery vehicles to promote transplanted hRPC migration and successful integration into degenerating retina.

The challenge in developing tissue-engineered bones (TEBs) for clinical applications lies in the constraints associated with the source and availability of autologous mesenchymal stem cells (MSCs) derived from the bone marrow, which creates a bottleneck. While allogeneic MSCs have shown promise in TEB applications, their ability to promote bone growth is notably diminished because of the inflammatory reaction at the transplant site and the inherent immune response triggered by allogeneic MSCs. Hence, there is a pressing need to develop methods that enhance the osteogenic differentiation of allogeneic MSCs during transplantation. Previous studies have found that IL-17 is a key proinflammatory factor in initiating inflammation and cascade amplification in the early stages of an inflammatory response, and proinflammatory cytokines such as TNF- and IL-17 can inhibit the osteogenic differentiation of MSCs in an immune environment. In this study, MSCs expressing HVEM were successfully constructed by viral transfection and further reconfirmed that IL-17 can inhibit the in vivo and in vitro osteogenesis of allogeneic MSCs through in vitro experiments and mouse calvarial bone defect (diameter about 3 mm) model, while MSCs that express herpesvirus-entry mediator (HVEM) exhibit the capacity to suppress immune responses and sustain strong osteogenic potential. We further pointed out that the mechanism by which HVEM promotes the osteogenesis of allogeneic MSCs is related to its inhibition of the IB kinase (IKK)-NF-B signaling pathway activated by IL-17 in the immune environment, which can significantly inhibit the ubiquitination and degradation of -catenin in MSCs induced by the IKK-NF-B pathway, upregulate the expression of -catenin, and promote bone formation. Hence, this research provides an initial connection between the Wnt/-catenin signaling pathway and the IKK-NF-B pathway during allogeneic MSC transplantation, offering new avenues for investigation and establishing a theoretical foundation for the potential use of HVEM-expressing MSCs in clinical treatments for bone defects.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are used to model cardiac development and disease. This requires a robust population of mature CMs and external stimuli to mimic the complex environment of the heart. In effort toward this maturation, previous groups have applied electrical stimulation (ES) to CMs with varying results depending on the stimulation duration, frequency, and pattern. As such, there is an uncertainty surrounding the timeline on which stimulated iPSC-CMs begin to show early signs of maturation in comparison with their nonstimulated counterparts. Here, we introduce a low-cost custom bioreactor capable of delivering tunable ES to standard 2D cell monolayers. We show that, after exposure to short-term ES, stimulated CMs express early signs of maturation compared to nonstimulated controls. Changes to contractility and protein expression indicate cellular rearrangement within cell monolayers and induction of partial maturation in response to ES. While early signs of maturation are present after 3-4 days of ES, additional cellular structures must develop to reach complete maturation. We also show that this bioreactor can electrically stimulate cardiac fibroblasts (cFBs) and may induce alignment of cFB. We have shown that our custom ES bioreactor can be easily integrated into standard in vitro cell culture platforms to induce measurable changes in both CMs and cFB, exhibiting its potential for promoting crucial CM maturation and cell alignment for cardiac tissue engineering applications.

Tissue engineering is a promising approach for the production of small-diameter vascular grafts; however, there are limited data directly comparing the suitability of applicable cell types for vessel biofabrication. Here, we investigated the potential of adult smooth muscle cells (SMCs), placental mesenchymal stem cells (MSCs), placental endothelial colony-forming cells (ECFCs), and a combination of MSCs and ECFCs on highly porous biocompatible poly(-caprolactone) (PCL) scaffolds produced via melt electrowriting (MEW) for the biofabrication of tissue-engineered vascular grafts (TEVGs). Cellular attachment, proliferation, and deposition of essential extracellular matrix (ECM) components were analysed over four weeks. TEVGs cultured with MSCs accumulated the highest levels of collagenous components within a dense ECM, while SMCs and the coculture were more sparsely populated, ascertained via histological and immunofluorescence imaging, and biochemical assessment. Scanning electron microscopy (SEM) enabled visualisation of morphological differences in cell attachment and growth, with MSCs and SMCs infiltrating and covering scaffolds completely within the 28-day culture period. Coverage and matrix deposition by ECFCs was limited. However, ECFCs lined the ECM formed by MSCs in coculture, visualised via immunostaining. Thus, of cells investigated, placental MSCs were identified as the preferred cell source for the fabrication of tissue-engineered constructs, exhibiting extensive population of porous polymer scaffolds and production of ECM components; with the inclusion of ECFCs for luminal endothelialisation, an encouraging outcome warranting further consideration in future studies. In combination, these findings represent a substantial step toward the development of the next generation of small-diameter vascular grafts in the management of cardiovascular disease.

Managing large, critical-sized bone defects poses a complex challenge, especially when autografts are impractical due to their size and limited availability. In such situations, the development of synthetic bone implants becomes crucial. These implants can be carefully designed and manufactured as potential bone substitutes, offering controlled parameters such as porosity, hardness, and osteogenic cues. In this study, we employed digital light processing (DLP) technology to construct an alumina ceramic scaffold featuring a triply periodic minimal surface (TPMS) structure for bone transplantation. The scaffold was filled with type I collagen to enhance cell infiltration [1], thereby increasing the total surface area. In addition, type I collagen is a carrier for both bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA). Using a clinically relevant rabbit cranium defect model, the scaffold underwent in vivo assessment for its functionality in repairing critical-sized bone defect (approximately 8 mm). Four groups of animal experiments were carried out including the control group, the gyroid scaffold group, the type I collagen-loaded scaffold group, and the bioactive factor-functionalized scaffold group. Our animal-based study results revealed that the gyroid scaffold, functionalized with bioactive molecules, provided a conductive surface for promoting increased bone formation and enhancing the healing process in critical-sized long bone and cranium defects. These findings offer preclinical evidence, supporting the use of a TPMS structure composite scaffold and present compelling support for its application as an advanced synthetic bone substitute in the future.

Persistent air leaks caused by thoracic surgery, physical trauma, or spontaneous pneumothoraces are a cause of patient morbidity with need for extended chest tube durations and surgical interventions. Current treatment measures involve mechanical closure of air leaks in the compromised pleura. Organ and membrane decellularisation offers a broad range of biomimetic scaffolds of allogeneic and xenogeneic origins, exhibiting innate tissue-specific characteristics. We explored a physicochemical method for decellularising porcine pleural membranes (PPM) as potential tissue-engineered surrogates for lung tissue repair. Decellularised PPM (dPPM) was characterised with histology, quantitative assays, mechanical testing, and sterility evaluation. Cytotoxicity and recellularisation assays assessed biocompatibility of decellularised PPM (dPPM). Haematoxylin and Eosin (H&E) staining showed an evident reduction in stained nuclei in the dPPM, confirmed with nuclear staining and analysis ( < 0.0001). Sulphated glycosaminoglycans (sGAG) and collagen histology demonstrated minimal disruption to the gross structural assembly of core extracellular matrix (ECM) in dPPM. Confocal imaging demonstrated realignment of ECM fibres in dPPM against native control. Quantitative analysis defined a significant change in the angular distribution ( < 0.0001) and coherence ( < 0.001) of fibre orientations in dPPM versus native ECM. DNA quantification indicated ≥85% reduction in native nuclear dsDNA in dPPM ( < 0.01). Collagen and sGAG quantification indicated reductions of both ( < 0.01). dPPM displayed increased membrane thickness ( < 0.001). However, Young's modulus (459.67 ± 10.36 kPa) and ultimate tensile strength (4036.22 ± 155.1 kPa) of dPPM were comparable with those of native controls at (465.82 ± 10.51 kPa) and (3912.9 ± 247.42 kPa), respectively. cytotoxicity and scaffold biocompatibility assays demonstrated robust human mesothelial cell line (MeT-5A) attachment and viability. DNA quantification in reseeded dPPM with MeT-5A cells exhibited significant increase in DNA content at day 7 ( < 0.01) and day 15 ( < 0.0001) against unseeded dPPM. Here, we define a decellularisation protocol for porcine pleura that represents a step forward in their potential tissue engineering applications as bioscaffolds.

The induced membrane (IM) preclinical models have been described in small animals, but few studies have looked at bone regeneration achievement. The optimisation and validation of such a preclinical model, considering the results obtained after the use of biomaterials as a substitute for bone grafting, could lead to simplifying the surgical procedure and enhance the clinical results. An in vivo model of the IM technique was developed on the femur of Lewis rats after a 4-mm critical bone defect stabilised with an osteosynthesis plate. A first optimisation phase was performed by evaluating different osteotomy methods and two different osteosynthesis plate sizes. The efficiency of the model was evaluated by the failure rate obtained 6 weeks after the first operative time. Thereafter, bone regeneration was evaluated histologically and radiologically at 24 weeks to confirm the critical nature of the bone defect (negative control), the effectiveness of the IM with a syngeneic bone graft (positive control) and the possibility of using a biomaterial (GlassBone Noraker) in this model. Sixty-three rats were included and underwent the first surgical step. Nineteen rats subsequently underwent the second surgical step. The results obtained led to select piezotomy as the best osteotomy technique and 1-mm-thick plates with 2.0-mm-diameter screws as osteosynthesis material. Twenty-four weeks after the second surgical step, solely the group with both surgical steps and a syngeneic bone graft showed complete ossification of the bone defect. In contrast, the group without a graft did not present a suitable ossification, which confirms the critical nature of the defect. IM produced an incomplete bone regeneration using GlassBone alone. A piezotome osteotomy with an osteosynthesis plate of sufficient stiffness is required for this two-stage bone regeneration model in rats. The 4-mm bone defect is critical for this model and suitable for biomaterial evaluation.

Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into adipocytes, chondrocytes, and osteoblasts. Owing to their differentiation potential, hMSCs are among the cells most frequently used for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, which necessitates expansion. Conventionally, this is often performed on rigid surfaces such as tissue culture plates (TCPs). However, during expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, rendering them unsuitable for clinical use. Although multiple approaches have been attempted to maintain hMSC stemness during prolonged expansion, finding a suitable culture system remains an unmet need. Recently, a few research groups have shown that hMSCs maintain their stemness over long passages when cultured on soft substrates. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. Furthermore, it has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay replicative senescence in hMSCs during long-term expansion on TCPs. However, the mechanism by which chromatin condensation/decondensation influences nuclear morphology and DNA damage, which are strongly related to the onset of senescence, remains unknown. To answer this question, we cultured hMSCs for long duration in the presence of epigenetic modifiers, histone acetyltransferase inhibitor (HATi), which promotes chromatin condensation by preventing histone acetylation, and histone deacetylase inhibitor (HDACi), which promotes chromatin decondensation, and investigated their effects on various nuclear markers related to senescence. We found that consistent acetylation causes severe nuclear abnormalities, whereas chromatin condensation by deacetylation helps to safeguard the nucleus from damage caused by expansion.

Cell macroencapsulation devices (CMD) offer a promising solution for organ function replacement by shielding implanted cells from the host immune system while allowing the exchange of nutrients and waste products. Developing efficient CMD necessitates optimizing vascular integration, membrane permeability, and cellular functionality using robust preclinical models. In this study, we adapted the chick chorioallantoic membrane (CAM) model to develop and evaluate CMD.