Providing a functional pericellular matrix (PCM) by fine-tuning the microenvironment of the articular chondrocytes (ACs) can greatly improve the outcomes of articular cartilage tissue engineering. While harvesting ACs with their PCM (chondrons) results in a low cell yield and a heterogeneous mixture of ACs and chondrons, microscale hydrogels could be used for mechanical tuning of the cell microenvironment. This may enable the use of stiffer bulk materials, improving the load-bearing capacity of the construct. This study investigates the effect of microenvironmental stiffness, independent of total construct stiffness, on the regenerative performance of ACs (ECM and PCM synthesis). Additionally, we explored articular cartilage-derived progenitor cells (ACPCs) as a possible alternative to ACs in the presented system. ACs were cultured in a soft or stiff bulk hydrogel (GelMA) or were encapsulated in soft microgels and seeded into the stiff GelMA. Constructs were seeded in an ex vivo porcine chondral defect model and cultured for 28 days with dynamic mechanical stimulation using a compression-sliding bioreactor. PCM and ECM quality were assessed through cell content analysis, immunofluorescent staining, histology, and measurements of GAG and collagen content. Cell encapsulation influenced ECM synthesis and PCM amount and completeness throughout the construct. Although the nonencapsulated groups showed stronger overall alcian blue staining, the encapsulated groups demonstrated more uniform matrix deposition throughout the depth of the tissue. Furthermore, ACPCs performed similarly to ACs. These findings suggest that the approach to differentially tune encapsulating and bulk hydrogel properties holds potential for future articular cartilage tissue engineering, and that ACPCs could be used as an alternative cell source.
Fibrosis and angiogenesis are key contributors to synovial inflammation in both the early and progressive stages of rheumatoid arthritis (RA) and osteoarthritis (OA), making them important therapeutic targets to mitigate joint tissue damage. In vitro drug screening, particularly for antifibrotic and antiangiogenic efficacy, is a standard method for evaluating therapeutic candidates prior to in vivo testing. Traditionally, most studies have relied on two-dimensional (2D) monolayer cell cultures, which lack physiologically relevant cell-matrix and cell-cell interactions. Substantial evidence now indicates that three-dimensional (3D) culture systems more accurately recapitulate the structural and functional complexity of native tissue environments. We employed 3D spheroid culture models of fibrosis and neovascularization to evaluate the antiangiogenic and antifibrotic effects of pirfenidone (PFD), an FDA-approved drug for idiopathic pulmonary fibrosis. Spheroid monocultures of 3T3 fibroblasts and co-cultures of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (SMCs) were encapsulated in cell-adhesive, proteolytically degradable polyethylene glycol (PEG) hydrogel scaffolds. The temporal effects of PFD dose and timing of addition in culture on fibroblast outgrowth, vascular sprouting, and viability were quantified up to 14 days. PFD treatment led to dose-dependent inhibition of both fibroblast outgrowth and vascular sprouting, depending on the initial timing of PFD addition, with cell viability maintained under all conditions. In addition, PFD reversed the onset of fibrosis and neovascularization. PFD exhibited antifibrotic activity and antiangiogenic potential in 3D cultures.
Objective:Reconstruction of critical-sized bone defects, particularly in the cranio-maxillofacial region, presents unique challenges due to the need for integration with adjacent well-vascularized tissue and the absence of significant load-bearing requirements. This study evaluated the clinical readiness of bone tissue engineering (BTE) for critically sized cranial defects using custom 3D-printed hydroxyapatite scaffolds augmented with either recombinant human bone morphogenetic protein-2 (rhBMP-2) or dipyridamole (DIPY) in a highly translational nonhuman primate model. Methods:Identical 5 × 5-cm vertex guided craniotomies were created in 12 macaques: Three cynomolgus macaques served as negative controls to validate the critical size nature of the defect, while nine rhesus macaques underwent scaffold reconstruction. Subjects were divided into three groups: uncoated scaffolds (n = 3), scaffolds augmented with rhBMP-2 (Infuse® Medtronic, n = 3), and scaffolds coated with DIPY, an adenosine A2A receptor (A2AR) indirect agonist (n = 3). Bone growth and integration were assessed over 12 months through serial CT scans, followed by ex vivo micro-CT scanning, histology, and nanoindentation testing. Results:Negative control subjects did not demonstrate new bone formation, confirming the critical defect model. Subjects treated with scaffolds through all treatment groups remained intact throughout the 12-month follow-up. The rhBMP-2-treated group exhibited bridging, ∼90% circumferentially, significantly greater than DIPY (∼9%) or the uncoated scaffold (10%) (p < 0.001). Bone volume within rhBMP-2-treated scaffolds (7621 ± 145 mm3) significantly exceeded that of DIPY (6466 ± 693 mm3, p = 0.03) and uncoated scaffold (6348 ± 663 mm3, p = 0.02) groups at 12 months. Quantitative histological micrograph analysis demonstrated that rhBMP-2 scaffolds were associated with the highest bone ingrowth (∼64%) relative to DIPY (∼39%) and uncoated scaffolds (∼27%). Nanoindentation yielded superior mechanical properties (Young's modulus and hardness) of newly generated bone with defects treated with rhBMP-2 scaffolds (p < 0.05). Conclusions:Reconstructing critically sized cranial defects with custom 3D-printed hydroxyapatite scaffolds was successful and yielded favorable results in this model. Scaffolds augmented with rhBMP-2 demonstrated superior bone ingrowth, integration, and mechanical properties, highlighting their potential as a viable alternative to autografts and allograft materials for cranioplasty.
Chronic respiratory diseases are a major global health concern. Lung epithelial dysfunction is a common underlying feature of many such conditions; hence, reconstructing the diseased epithelium with functional epithelial cells is a promising therapeutic approach. There are various endogenous stem cell and progenitor populations in the lung epithelium that can be utilized for transplantation. Additionally, pluripotent stem cells (PSCs) have emerged as a valuable source for generating therapeutic cells due to their capacity for indefinite self-renewal and the availability of directed differentiation protocols to transform them into lung progenitors or mature lung epithelial cells. This review discusses the endogenous stem cell and progenitor populations of the lung epithelium, recent advances in developing directed differentiation protocols to generate these cells, and the application of both endogenous and PSC-derived lung epithelial cells for disease modeling in vitro and as cell therapies in vivo. It provides valuable insights into the current progress of regenerative medicine within the respiratory field and highlights areas that require further research.
Meniscal injuries present a significant clinical challenge due to the limited self-healing capacity of avascular regions and the unsatisfactory long-term outcomes of current repair strategies. Collagen, the primary structural component of the meniscal extracellular matrix (ECM), plays a crucial role in maintaining its biomechanical integrity and guiding tissue regeneration. This review summarizes recent advances in collagen scaffolds technology, focusing on materials, collagen extraction, and scaffold fabrication methods, as well as their in vivo interactions with cells that regulate tissue regeneration. The mechanical enhancement of collagen scaffolds through crosslinking and reinforcement with synthetic polymers is discussed, alongside strategies for controlled degradation and biological integration. Despite remaining challenges in mechanical durability and long-term stability, these developments position collagen-based scaffolds as a promising avenue toward clinically viable meniscal repair solutions.
Decellularized articular cartilage of human origin presents itself as the most homologous filling material for focal cartilage defects. Yet, the full repopulation of the exceptionally dense collagen construct has never been achieved without providing host cells with artificially created migration paths into the matrix. Within this study, we examine the use of a femtosecond laser to engrave fine patterns into human articular cartilage before decellularization and GAG depletion (decell-deGAG). Scaffolds were tested for decellularization success and mechanical behavior. Seeding tests were performed to assess biocompatibility and examine the performance in a simulated defect environment using an osteochondral plug model in vitro and in vivo in an ectopic nude mouse model. The composition and structure of the newly formed repair tissue and macrophage recruitment were observed via histology. The femtosecond laser was successful in engraving deep, fine structures into the matrix without the thermal damage found with other laser techniques. Engraving was also beneficial for decellularization success. The resulting decell-deGAG scaffold featured a compressive modulus many times stronger than other biomaterials commonly used for cartilage regeneration and presents a defect filling material that is similar to the tissue it is meant to replace. Moreover, the incisions promoted the repopulation with therapeutically relevant cells. A favorable spatial environment inside the incisions facilitated the formation of repair tissue that mimics hyaline cartilage in composition and collagen orientation. Scaffolds were well-integrated within simulated defects. Femtosecond laser-engraved cartilage poses an authentic defect filling material with cartilage-like properties. When used in combination with cell seeding, it promotes the formation of differentiated repair tissue. Thus, the hereby presented biomaterial shows great potential in improving the repair of focal cartilage defects and reducing long-term graft failures.
The dermal barrier is widely considered the body's first line of defense against most foreign bodies, protecting it from both moisture loss and bacterial invasion. However, when the skin is ruptured for long-term medical interventions (e.g., transcutaneous prosthetics), it is difficult to restore and maintain this protective barrier. Although there are no direct, biological examples of true transcutaneous features in the human body, similar phenomena can be observed in phalangeal nails. This study aims to investigate keratin, the primary component of fingernails, in its hydrolyzed form as an additive to induce cell adhesion in two representative scaffold types. Electrospun fibers and chitosan-gelatin cryogels-two well-characterized scaffolds used in dermal tissue engineering-were selected for this study as a fibrous and macroporous foundation. Both electrospun fibers and cryogels were fabricated with a range of keratin additive concentrations (0, 1, 3, 5, 7, and 10 wt/wt% and wt/v% for electrospun fibers and cryogels, respectively) and tested for surface properties, mechanical strength, biocompatibility, and material behavior. Overall, it was determined that hydrolyzed keratin had a positive effect on cell adhesion and proliferation but that high quantities of the keratin resulted in adverse effects on the scaffold properties. With dermal applications in mind, this study found that 5 and 7 wt/wt% keratin electrospun fibers possessed required cell counts, surface energies, tensile strength, and contact angle, all with consistent reproducibility. For the cryogels, 3 and 5 wt/v% keratin had the best combined performance, maintained structural integrity through swelling and porosity, and displayed minimal loss in compressive strength. Therefore, hydrolyzed keratin represents a promising additive for bothelectrospun fibers and cryogels in tissue engineering applications.
Aims:The aim of this study is to investigate the effects of islet cells and mesenchymal stem cells transferred together in the amniotic membrane (AM) in order to preserve the viability and functionality of islet cells on the success of islet transplantation in diabetes mellitus-induced rats. Methods:A total of 80 male Wistar albino rats, aged 3.5-4 months, were included in this study. While 40 Wistar Albino rats were used for the process of islet cell isolation, 40 Wistar Albino rats were used to establish experimental groups. These rats were assigned to five experimental groups including eight rats in each. These groups were AM, amniotic membrane + mesenchymal stem cell (AM + MSC), amniotic membrane + islet cell (AM + IC), amniotic membrane + islet cell + mesenchymal stem cell (AM + IC + MSC), and sham groups. The study was concluded for 28 days. Results:Although there was no significant difference between AM + IC and AM + IC + MSC groups in terms of mean blood glucose levels, both groups had statistically different values compared to the sham group. A significant difference was observed between the AM + IC and AM + IC + MSC groups in the c-peptide levels before and after transplantation. Immunohistochemical staining illustrated the presence of insulin-positive cells in both AM + IC and AM + IC + MSC groups. Moreover, BrDU (+) cells were determined in AM + IC and AM + IC + MSC groups using BrDU staining. Conclusion:The study results indicated that transplanting islet cells into the omentum by being packaged in AM preserved their viability and function, leading to significant effects on blood glucose and c-peptide levels.
Glycolysis supports mesenchymal stem cell (MSC) proliferation and sustains their undifferentiated state by maintaining energy supply and limiting apoptosis. The rapid advancement of space life sciences has spurred considerable interest in the effects of microgravity on stem cells. However, the contribution of glycolytic metabolism to apoptotic regulation under simulated microgravity (SMG) remains unclear. This study examined the influence of SMG on glycolytic activity and apoptosis in human dental pulp stem cells (hDPSCs). Lactic acid and glucose measurements were used to evaluate glycolytic flux, while transcript levels of HK2, PKM2, and LDHA were quantified by qPCR, HK2 and PKM2 protein expression was assessed by Western blotting, and annexin V-FITC/PI staining combined with immunoblotting of apoptosis-related proteins (BAX, BCL-2, and cleaved caspase-3) was performed to assess cell death. SMG markedly increased glycolytic capacity and attenuated apoptosis in hDPSCs. SphK1 expression was also elevated, indicating a role in cell survival. Pharmacological inhibition of SphK1 with PF-543 reduced both glycolysis and the antiapoptotic effect, implicating SphK1 as a critical regulator of these processes. Inhibition of glycolysis by 2-DG further increased apoptosis, confirming the protective role of glycolytic metabolism under SMG. These findings demonstrate that SMG enhances glycolysis and limits apoptosis in hDPSCs via SphK1 upregulation, suggesting that microgravity conditions may augment stem cell survival and function.
Background: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. Methods:Semipermeable membranes were integrated into the CAM, with vascularization modulated through growth factors and extracellular matrix manipulation. Human kidney tubular epithelial cells were cultured on these vascularized membranes to assess cell viability, polarization, and functionality, including selective transport and barrier integrity. Results:The membranes integrated successfully into the CAM and supported functional vascularization, demonstrating selective permeability by facilitating the exchange of low-molecular-weight compounds while preventing the infiltration of larger proteins and cells, thereby creating an immune-isolated environment. Kidney tubular epithelial cells remained viable, polarized, and functionally active, showcasing selective compound transport and robust barrier integrity. Conclusion:These findings underscore the CAM model's utility in evaluating vascular integration, membrane permeability, and epithelial cell functionality, all critical parameters for CMD development. The CAM model provides a rapid, cost-effective platform for CMD assessment, significantly accelerating their development and potential clinical translation. This approach holds particular promise for applications targeting kidney diseases characterized by compromised transport functions, offering a pathway toward more effective therapeutic solutions.
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.
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.
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.
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.
Background: 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. Methods: 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. Results: 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. Conclusions: 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.
Background: Tendon injuries are increasingly common and heal by fibrosis rather than scar-less regeneration. Tissue engineering seeks to improve repair using synthetic polymer scaffolds with biomimetic factors to enhance the regenerative potential. Methods: In this study, we compared three groups, namely, poly(lactic acid) (PLA) meltblown scaffolds, PLA meltblown scaffolds coated with tendon-derived matrix (TDM), and PLA meltblown scaffolds with carbodiimide crosslinked TDM (2.5:1:1 EDC:NHS:COOH ratio) (EDC-TDM) and determined their potential for engineered tendon development. We cultured human adipose stem cells (hASCs) for 28 days on meltblown scaffolds (n = 4-6/group) and measured tensile mechanical function, matrix synthesis, and matrix composition using biochemical assays and proteomics. Results: Coating PLA meltblown scaffolds with TDM improved yield stretch and stress at 28 days compared with PLA. Matrix synthesis rates for TDM or EDC-TDM were similar to PLA. Proteomic analysis revealed that hASCs produced a collagen-rich extracellular matrix, with many tendon-related matrix proteins. Coating scaffolds with TDM led to an increase in collagen type I whereas EDC-TDM scaffolds had an increase in glycoproteins and ECM regulators compared with other groups, consistent with increased maturity of the newly deposited matrix. Conclusions: TDM coating and crosslinking of meltblown scaffolds demonstrated matricellular benefits for the proteome of engineered tendon development but provided fewer clear benefits toward mechanical, biochemical, and rate of matrix accumulation than expected, and that previous work with electrospun scaffolds would suggest. However, electrospun scaffolds have different fiber structure and microarchitecture than meltblown, suggesting that further consideration of these differences and refinement of TDM application methods to meltblown scaffolds is required.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) are well established for their osteogenic potential but are prone to senescence with aging or in vitro expansion. Drug treatments that reduce cellular senescence may enhance the regenerative capacity of BM-MSCs. This study investigates the effects of losartan and fisetin, both separately and in combination, on cellular senescence and osteogenesis. Human BM-MSCs were exposed to low and high concentrations of each drug for 24 h. Our findings showed that high-dose losartan exhibited cytotoxicity, focusing subsequent analyses on the low doses. Both low-dose losartan and fisetin effectively mitigated cellular senescence, with combined treatment showing synergistic effects in reducing senescence markers. From these initial findings, subsequent experiments utilized low doses of both compounds to evaluate their effect on differentiation capacity. Our multimodal approach, incorporating flow cytometry, senescence-associated heterochromatin foci (SAHF) immunohistochemistry, senescence-associated secretory phenotype (SASP) quantification, and differentiation potential assays, revealed that the combination of 23.6 μM of losartan and 50 μM of fisetin was optimal for reducing cellular senescence and enhancing osteogenesis in BM-MSCs. These results support potential therapeutic strategies to counteract age-related declines in bone health and improve healing. By targeting cellular senescence while promoting osteogenesis, losartan and fisetin offer promising avenues for future research aimed at enhancing the regenerative capacity of BM-MSCs in the context of musculoskeletal regenerative medicine.
