Tissue-engineered Constructs Research Articles

Multiphoton Imaging of Maturation in Tissue Engineering.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

Electromagnetic field-mediated chitosan/gelatin/nano-hydroxyapatite and bone-derived scaffolds regulate the osteoblastic and chondrogenic phenotypes of adipose-derived stem cells to construct osteochondral tissue engineering niche in vitro

It is critical to explore the effects of electromagnetic field (EMF) on the construction of functional osteochondral tissue, which has shown certain clinical significance for the treatment of osteochondral injury. At present, there are few studies on the effect of the direction of EMF on cells. This study aimed to investigate the effects of EMF coupling on different parameters to control adipose-derived stem cells (ADSCs) proliferation and specific chondrogenic and osteogenic differentiation at 2D level and 3D level. The proliferation and differentiation of EMF-induced ADSCs are jointly regulated by EMF and space structure. In this study, Cs7/Gel3/nHAP scaffolds were prepared with good degradation rate (86.75 ± 4.96 %) and absorb water (1100 %), and the pore size was 195.63 ± 54.72 μm. The bone-derived scaffold with a pore size of 267.17 ± 129.18 μm was obtained and its main component was hydroxyapatite. Cs7/Gel3/nHAP scaffolds and bone-derived scaffolds are suitable as 3D level materials. The optimal EMF intensity was 2 mT for chondrogenic differentiation and proliferation and 1 mT for osteogenic differentiation and proliferation. It is noteworthy that EMF has a negative correlation with ADSCs proliferation in the vertical direction at 2D level, while it has a positive correlation with ADSCs proliferation at 3D level. EMF mediated 3D osteochondral scaffold provide good strategy for osteochondral tissue engineering construction.

Bone defect management with tissue-engineered constructs based on deproteinized cancellous bone: an experimental study

Background Management of bone defects with autologous bone grafting has always been the "gold standard" but it is not always possible to use it for a number of reasons. Preprocessed materials of biological and non-biological origin were developed as an alternative. A new branch of these materials is tissue-engineered constructs that fully imitate autologous bone in required volume.Aim is to study in vivo the possibility of using deproteinized human cancellous bone tissue as a matrix for creating tissue-engineered constructs.Methods The study was carried out on 24 NZW line rabbits, since this line has a fully characterized stromal-vascular fraction formula (SVF). The study design included 3 groups. First group (control) had surgical modeling of bone defects in the diaphysis of the contralateral femur without reconstruction; Group 2 had bone defect reconstruction using fragments of a deproteinized cancellous bone graft; group 3 underwent bone defect reconstruction using fragments of deproteinized cancellous bone matrix along with the autologous adipose tissue SVF (obtained according to ACP SVF technology). Animals were sacrificed with ether anesthesia at 2, 4 and 6 weeks after the operation and subsequent histological study followed.Result During all periods of the study, the newly formed bone tissue volume density in the 3rd group (reconstruction with deproteinized human cancellous bone + stromal-vascular fraction) was 1.78 times higher (p < 0.001) than in the first group (bone defect without reconstruction), 1.21 times higher (p < 0.001) than in the 2nd group (reconstruction with deproteinized cancellous bone alone). The dynamics of changes in the mature bone tissue volume density was similar to those of the newly formed bone tissue.Discussion The comparative analysis of reparative processes using a tissue engeneered construst based on deproteinized cancellous human bone with adipose tissue stromal vascular fraction revealed that the use of these bone substitute materials contributes not only to the early activation of reparative regeneration of the main structural elements of bone tissue at the site of bone defect, but also their timely differentiation.Conclusion The use of deproteinized cancellous bone matrix combined with stromal-vascular fraction to create a tissue-engineered construct could unleash several regeneration mechanisms and accelerate the process of bone defect site repair, compared with 1st and 2nd group of study.

Combination of melt-electrospun poly-ε-caprolactone scaffolds and hepatocyte-like cells from footprint-free hiPSCs to create 3D biohybrid constructs for liver tissue engineering

The liver is a vital organ with numerous functions, including metabolic functions, detoxification, and the synthesis of secretory proteins. The increasing prevalence of liver diseases requires the development of effective treatments, models, and regenerative approaches. The field of liver tissue engineering represents a significant advance in overcoming these challenges. In this study, 3D biohybrid constructs were created by combining hepatocyte-like cells (HLCs) derived from patient-specific footprint-free human induced pluripotent stem cells (hiPSCs) and 3D melt-electrospun poly-ε-caprolactone (PCL) scaffolds. First, a differentiation procedure was established to obtain autologous HCLs from hiPSCs reprogrammed from renal epithelial cells using self-replicating mRNA. The obtained cells expressed hepatocyte-specific markers and exhibited important hepatocyte functions, such as albumin synthesis, cytochrome P450 activity, glycogen storage, and indocyanine green metabolism. Biocompatible PCL scaffolds were fabricated by melt-electrospinning and seeded with pre-differentiated hepatoblasts, which uniformly attached to the fibers of the scaffolds and successfully matured into HLCs. The use of patient-specific, footprint-free hiPSC-derived HLCs represents a promising cell source for personalized liver regeneration strategies. In combination with biocompatible 3D scaffolds, this innovative approach has a broader range of applications spanning liver tissue engineering, drug testing and discovery, and disease modeling.

Restoration of X-ray bone density when replacing cortical plate defects with a tissue-engineered construct in the experiment

Over the past decade, in global practice, the frequency of using high-resolution multi-layer spiral computed tomography (MSCT) for assessing the state of cancellous and cortical bone tissue has significantly increased. Using high-resolution MSCT makes it possible to assess X-ray bone density at various times after replacement of cortical plate defects with osteoplastic materials. The aim of the research. To study the restoration of cortical bone density in the area of osteoplasty using tissue-engineered construct in the experiment. Materials and methods. In an in vivo experiment on New Zeland White (NZW) rabbits, perforation defects of cortical bone were formed in the femoral diaphysis. Three study groups were set up: group 1 – without bone defect replacement; group 2 – with bone defect replacement with deproteinized cancellous bone; group 3 – with bone defect replacement with tissue-engineered construct based on deproteinized cancellous bone with stromal vascular fraction of adipose tissue. Follow-up periods were 2, 4 and 6 weeks after the surgery. The X-ray density of cortical bone tissue was measured in Hounsfield units (HU). Fragments of deproteinized human cancellous bone were used alone and in combination with the stromal vascular fraction of NZW rabbit adipose tissue as a bone-replacing material for bone defect replacement. Results. Cortical plate density the in the area of the defect in the group 3 by the week 6 is on average 1.3 times lower than that of the intact cortical plate and corresponds to D1 according to Misch classification. Cortical plate density in the area of the defect on the side of medullary canal by the week 6 in the group 3 corresponds to D1 according to Misch classification and is equal to 1351.25 ± 221.18 HU (1052; 1805), which is 1.5 times higher than in group 2 (D2 according to Misch classification; p < 0.05). The obtained results indicate an earlier restoration of X-ray bone density when using a tissue-engineered construct (group 3) compared to the same indicators in groups 1 and 2.

Development of a testing machine for biaxial testing of soft tissue and biomaterials

Knowledge of the mechanical properties of soft tissues and biomaterials is the keystone for predictive modeling in cardio- and endovascular surgery, traumatology and orthopedics, neurosurgery, and the development of tissue-engineering constructs. Needed mechanical properties can be only explored via experimental tests. Therefore, mechanical experiments are forming the foundation of medical decision support systems, which are impossible without adequate mathematical models of the processes involved. Sirius University of Science and Technology has launched the project "Experimental research and mathematical modeling of the mechanical behavior of biomaterials". The goal of the project is to create an infrastructure for experimental study of the mechanics of soft tissues and biomaterials. At this stage, a machine for biaxial testing of soft tissues and biomaterials has been developed and put into trial operation. The developed machine takes into account the features of working with soft tissues: small sample size, maintaining certain environmental conditions to preserve the sample, various types of sample gripping to minimize edge effects and no slippage, non-contact methods for assessing displacement and deformation fields, variability in sample loading protocols. The result of the test is a data set that includes a test report and synchronized output readings of strain gauge, temperature, optoelectronic sensors with high-resolution images. Uniaxial verification tests of acrylic tapes VHB4910 were carried out by the developed machine. The results were compared with data obtained on a commercial dynamic mechanical analyzer RSA-G2 (TA Instruments) for similar materials under the same test conditions. A series of biaxial cyclic experiments were carried out for the pericardium of cattle, the cardiovascular patch Ecofolon PS04/PS01 (JSC NPK Ecoflon, St. Petersburg, Russia), and the GoreTex vascular graft (W.L. Gore and Associates, USA).

Perforated red blood cells enable compressible and injectable hydrogels as therapeutic vehicles

Hydrogels engineered for medical use within the human body need to be delivered in a minimally invasive fashion without altering their biochemical and mechanical properties to maximize their therapeutic outcomes. In this regard, key strategies applied for creating such medical hydrogels include formulating precursor solutions that can be crosslinked in situ with physical or chemical cues following their delivery or forming macroporous hydrogels at sub-zero temperatures via cryogelation prior to their delivery. Here, we present a new class of injectable composite materials with shape recovery ability. The shape recovery is derived from the physical properties of red blood cells (RBCs) that are first modified via hypotonic swelling and then integrated into the hydrogel scaffolds before polymerization. The RBCs’ hypotonic swelling induces the formation of nanometer-sized pores on their cell membranes, which enable fast liquid release under compression. The resulting biocomposite hydrogel scaffolds display high deformability and shape-recovery ability. The scaffolds can repeatedly compress up to ∼87% of their original volumes during injection and subsequent retraction through syringe needles of different sizes; this cycle of injection and retraction can be repeated up to ten times without causing any substantial mechanical damage to the scaffolds. Our biocomposite material system and fabrication approach for injectable materials will be foundational for the minimally invasive delivery of drug-loaded scaffolds, tissue-engineered constructs, and personalized medical platforms that could be administered to the human body with conventional needle-syringe systems.

From Cultured Chats to the Chirrups of Choo-Choo-Da-Choos, or How We Found a Key to the Gate of Eden

Reinvention of the form of expression is a conceptual approach characteristic for the evolution of all arts. This research study provides one such step forward in the advancement of scientific paper, a standard form of expression in natural sciences, toward more progressive terrains. The paper adopts the form of a theatrical play where a scientific family of four attempts to find the way around a writer’s block (Act I). Their idealess sense of confinement is overcome through arts or, more specifically, through scientific research implementing the aesthetics of music as its key element. The characters record compositions differing in the degrees of consonance, rhythmicity and tonality, and play them to birds to observe their vocal and behavioral responses (Act II). They note that the birdsong volume drops significantly when the piano tunes, especially the compositionally complex ones, are played to the birds and increases signifcantly when the white noise of ocean waves is played. The bird count experiment proves indirectly that the birdsong volume reduction is an auditory response of the birds to music rather than the effect of their migrating to or away from the sound source. This science with the flavor of art elevates the characters beyond the limits of their confinement and they, exalted and uplifted, gain ideas for creative research in their own disciplines of biomedical science (Act III). They relinquish the newly gained freedom in favor of embracing love for humanity that underlies medical research and return to their confinement, where they conduct, in the coda of the paper, a series of experiments that venture beyond musicology and ornithology and fall in the domains of tissue engineering and drug delivery, but also psychology (Act III). First, sparrow nests are used as templates for tissue engineering constructs, which facilitate the growth of human fibroblastic cells better than their artificial, gridded replicas. Next, the shell of an Easter egg, predominantly calcite in composition, is used with success as a drug delivery carrier. Lastly, the bird nest drawing experiment demonstrates a greater mental health of children than that of the adults, correlating with one of the overarching points of the paper, which is to present children as a model for grownups to follow rather than the other way around. Over the course of the play, the characters discuss the value of the various fusions between art and science, question the limits of human language and repeatedly go off-topic to celebrate the freeness of the creative thinking process. The play reports on real-life research methods and scientific results through a narrative, without diminishing their empirical rigor and analytical accuracy.

From Cultured Chats to the Chirrups of Choo-Choo-Da-Choos, or How We Found a Key to the Gate of Eden

Reinvention of the form of expression is a conceptual approach characteristic for the evolution of all arts. This research study provides one such step forward in the advancement of scientific paper, a standard form of expression in natural sciences, toward more progressive terrains. The paper adopts the form of a theatrical play where a scientific family of four attempts to find the way around a writer’s block (Act I). Their idealess sense of confinement is overcome through arts or, more specifically, through scientific research implementing the aesthetics of music as its key element. The characters record compositions differing in the degrees of consonance, rhythmicity and tonality, and play them to birds to observe their vocal and behavioral responses (Act II). They note that the birdsong volume drops significantly when the piano tunes, especially the compositionally complex ones, are played to the birds and increases signifcantly when the white noise of ocean waves is played. The bird count experiment proves indirectly that the birdsong volume reduction is an auditory response of the birds to music rather than the effect of their migrating to or away from the sound source. This science with the flavor of art elevates the characters beyond the limits of their confinement and they, exalted and uplifted, gain ideas for creative research in their own disciplines of biomedical science (Act III). They relinquish the newly gained freedom in favor of embracing love for humanity that underlies medical research and return to their confinement, where they conduct, in the coda of the paper, a series of experiments that venture beyond musicology and ornithology and fall in the domains of tissue engineering and drug delivery, but also psychology (Act III). First, sparrow nests are used as templates for tissue engineering constructs, which facilitate the growth of human fibroblastic cells better than their artificial, gridded replicas. Next, the shell of an Easter egg, predominantly calcite in composition, is used with success as a drug delivery carrier. Lastly, the bird nest drawing experiment demonstrates a greater mental health of children than that of the adults, correlating with one of the overarching points of the paper, which is to present children as a model for grownups to follow rather than the other way around. Over the course of the play, the characters discuss the value of the various fusions between art and science, question the limits of human language and repeatedly go off-topic to celebrate the freeness of the creative thinking process. The play reports on real-life research methods and scientific results through a narrative, without diminishing their empirical rigor and analytical accuracy.

Experimental replacement of various bladder volumes with allogeneic tissue-engineered constructions

The results of experimental replacement of the bladder wall up to subtotal using multicomponent tissue-engineered structures are presented. Purpose. Development and experimental use of a tissue-engineered structure for replacing various volumes of the bladder wall. Material and methods. The original poly-L,L-lactide matrix is reinforced with silk fibroin. Mesenchymal cells were introduced into the constructs. 6 intact animals underwent filling cystometry. The maximum cystometric capacity was 11.2±0.97 ml. In these same 6 animals, the anesthetic capacity of the bladder was measured, which was 23.83±0.71 ml. 36 animals underwent reconstruction of the bladder using a prepared tissue-engineered construct after resection of the corresponding volume of the organ. Groups of 9 animals received bladder volumes of 5, 10, 15 and 20 ml. The observation period was 3 months. Results: According to computed tomography of the abdominal and pelvic organs (native study and with intravesical administration of a radiocontrast agent), 4, 8, 12 weeks after surgery, a bladder of physiological capacity is determined in all study groups, the implanted structure is visualized as a hyperintense signal in area of the apex of the bladder. no leakage of contrast agent is detected. Filling cystometry in 2 animals that underwent replacement of 20 ml of bladder volume (subtotal replacement) after 12 weeks showed that the capacity of the formed reservoir correlates with preoperative parameters. Macroscopically, the anastomosis zone is consistent in all groups of animals, the tissue-engineered structure is determined at the implantation site, lysis of the structure is noted by 12 weeks of observation with the preservation of small residual fragments at the implantation site. Conclusion. The experimental use of the developed tissue-engineered multicomponent structure turned out to be effective for replacing defects of the bladder wall of various volumes up to subtotal reconstruction. Further study of technologies for the use of tissue-engineered allogeneic constructs can significantly improve the results of treatment of urological pathologies for which obtaining autologous material is not possible.

Examining the Potential of Vitamin C Supplementation in Tissue-Engineered Equine Superficial Digital Flexor Tendon Constructs.

Because equine tendinopathies are slow to heal and often recur, therapeutic strategies are being considered that aid tendon repair. Given the success of utilizing vitamin C to promote tenogenesis in other species, we hypothesized that vitamin C supplementation would produce dose-dependent improvements in the tenogenic properties of tendon proper (TP) and peritenon (PERI) cells of the equine superficial digital flexor tendon (SDFT). Equine TP- and PERI-progenitor-cell-seeded fibrin three-dimensional constructs were supplemented with four concentrations of vitamin C. The gene expression profiles of the constructs were assessed with 3'-Tag-Seq and real-time quantitative polymerase chain reaction (RT-qPCR); collagen content and fibril ultrastructure were also analyzed. Moreover, cells were challenged with dexamethasone to determine the levels of cytoprotection afforded by vitamin C. Expression profiling demonstrated that vitamin C had an anti-inflammatory effect on TP and PERI cell constructs. Moreover, vitamin C supplementation mitigated the degenerative pathways seen in tendinopathy and increased collagen content in tendon constructs. When challenged with dexamethasone in two-dimensional culture, vitamin C had a cytoprotective effect for TP cells but not necessarily for PERI cells. Future studies will explore the effects of vitamin C on these cells during inflammation and within the tendon niche in vivo.

Multimaterial 3D and 4D Bioprinting of Heterogenous Constructs for Tissue Engineering.

Additive manufacturing (AM), which is based on the principle of layer-by-layer shaping and stacking of discrete materials, has shown significant benefits in the fabrication of complicated implants for tissue engineering (TE). However, many native tissues exhibit anisotropic heterogenous constructs with diverse components and functions. Consequently, the replication of complicated biomimetic constructs using conventional AM processes based on a single material is challenging. Multimaterial 3D and 4D bioprinting (with time as the fourth dimension) has emerged as a promising solution for constructing multifunctional implants with heterogenous constructs that can mimic the host microenvironment better than single-material alternatives. Notably, 4D-printed multimaterial implants with biomimetic heterogenous architectures can provide a time-dependent programmable dynamic microenvironment that can promote cell activity and tissue regeneration in response to external stimuli. This paper first presents the typical design strategies of biomimetic heterogenous constructs in TE applications. Subsequently, the latest processes in the multimaterial 3D and 4D bioprinting of heterogenous tissue constructs are discussed, along with their advantages and challenges. In particular, the potential of multimaterial 4D bioprinting of smart multifunctional tissue constructs is highlighted. Furthermore, this review provides insights into how multimaterial 3D and 4D bioprinting can facilitate the realization of next-generation TE applications.

Ascorbic Acid Enhances the Metabolic Activity, Growth and Collagen Production of Human Dermal Fibroblasts Growing in Three-dimensional (3D) Culture

Tissue engineering (TE) enables the development of functional synthetic substitutes to be replaced with damaged tissues and organs instead of the use of auto or allografts. A wide range of biomaterials is currently in use as TE scaffolds. Among these materials, naturally sourced ones are favorable due to being highly biocompatible and supporting cell growth and function, whereas synthetic ones are advantageous because of the high tunability on mechanical and physical properties as well as being easy to process. Alongside the advantages of synthetic polymers, they mostly show hydrophobic behavior that limits biomaterial-cell interaction and, consequently, the functioning of the developed TE constructs. In this study, we assessed the impact of L-Ascorbic acid 2-phosphate (AA2P) on improving the culture conditions of human dermal fibroblasts (HDFs) growing on a three-dimensional (3D) scaffold made of polycaprolactone (PCL) using emulsion templating. Our results demonstrated that AA2P enhances the metabolic activity and growth of HDFs as well as collagen deposition by them when supplemented in their growth medium at 50 µg/mL concentration. It showed a great potential to be used as a growth medium supplement to circumvent the disadvantages of culturing human cells on a synthetic biomaterial that is not favored in default. AA2P's potential to improve cell growth and collagen deposition may prove an effective way to culture human cells on 3D PCL PolyHIPE scaffolds for various TE applications.

Non-neotissue constituents as underestimated confounders in the assessment of tissue engineered constructs by near-infrared spectroscopy

Non-destructive assessments are required for the quality control of tissue-engineered constructs and the optimization of the tissue culture process. Near-infrared (NIR) spectroscopy coupled with machine learning (ML) provides a promising approach for such assessment. However, due to its nonspecific nature, each spectrum incorporates information on both neotissue and non-neotissue constituents of the construct; the effect of these constituents on the NIR-based assessments of tissue-engineered constructs has been overlooked in previous studies. This study investigates the effect of scaffolds, growth factors, and buffers on NIR-based assessments of tissue-engineered constructs. To determine if these non-neotissue constituents have a measurable effect on the NIR spectra of the constructs that can introduce bias in their assessment, nine ML algorithms were evaluated in classifying the NIR spectra of engineered cartilage according to the scaffold used to prepare the constructs, the growth factors added to the culture media, and the buffers used for storing the constructs. The effect of controlling for these constituents was also evaluated using controlled and uncontrolled NIR-based ML models for predicting tissue maturity as an example of neotissue-related properties of interest. Samples used in this study were prepared using norbornene-modified hyaluronic acid scaffolds with or without the conjugation of an N-cadherin mimetic peptide. Selected samples were supplemented with transforming growth factor-beta1 or bone morphogenetic protein-9 growth factor. Some samples were frozen in cell lysis buffer, while the remaining samples were frozen in PBS until required for NIR analysis. The ML models for classifying the spectra of the constructs according to the four constituents exhibited high to fair performances, with F1 scores ranging from 0.9 to 0.52. Moreover, controlling for the four constituents significantly improved the performance of the models for predicting tissue maturity, with improvement in F1 scores ranging from 0.09 to 0.77. In conclusion, non-neotissue constituents have measurable effects on the NIR spectra of tissue-engineered constructs that can be detected by ML algorithms and introduce bias in the assessment of the constructs by NIR spectroscopy. Therefore, controlling for these constituents is necessary for reliable NIR-based assessments of tissue-engineered constructs.

Post-Implantation Inflammatory Responses to Xenogeneic Tissue-Engineered Cartilage Implanted in Rabbit Trachea: The Role of Cultured Chondrocytes in the Modification of Inflammation

Immune responses to tissue-engineered grafts made of xenogeneic materials remain poorly studied. The scope of current investigations is limited by the lack of information on orthotopically implanted grafts. A deeper understanding of these processes is of great importance since innovative surgical approaches include the implantation of xenogeneic decellularized scaffolds seeded by cells. The purpose of our work is to study the immunological features of tracheal repair during the implantation of tissue-engineered constructs based on human xenogeneic scaffolds modified via laser radiation in rabbits. The samples were stained with hematoxylin and Safranin O, and they were immunostained with antibodies against tryptase, collagen II, vimentin, and CD34. Immunological and inflammatory responses were studied by counting immune cells and evaluating blood vessels and collagen. Leukocyte-based inflammation prevailed during the implantation of decellularized unseeded scaffolds; meanwhile, plasma cells were significantly more abundant in tissue-engineered constructs. Mast cells were insignificantly more abundant in tissue-engineered construct samples. Conclusions: The seeding of decellularized xenogeneic cartilage with chondrocytes resulted in a change in immunological reactions upon implantation, and it was associated with plasma cell infiltration. Tissue-engineered grafts widely differed in design, including the type of used cells. The question of immunological response depending on the tissue-engineered graft composition requires further investigation.

Microscale strain concentrations in tissue-engineered osteochondral implants are dictated by local compositional thresholds and architecture

Tissue-engineered osteochondral implants manufactured from condensed mesenchymal stem cell bodies have shown promise for treating focal cartilage defects. Notably, such manufacturing techniques have shown to successfully recapture the bulk mechanical properties of native cartilage. However, the relationships among the architectural features, local composition, and micromechanical environment within tissue-engineered cartilage from cell-based aggregates remain unclear. Understanding such relationships is crucial for identifying critical parameters that can predict in vivo performance. Therefore, this study investigated the relationship among architectural features, composition, and micromechanical behavior of tissue-engineered osteochondral implants. We utilized fast-confocal microscopy combined with a strain mapping technique to analyze the micromechanical behavior under quasi-static loading, as well as Fourier Transform Infrared Spectroscopy to analyze the local compositions. More specifically, we investigated the architectural features and compositional distributions generated from tissue maturation, along with macro- and micro-level strain distributions. Our results showed that under compression, cell-based aggregates underwent deformation followed by body movement, generating high local strain around the boundaries, where local aggrecan concentration was low and local collagen concentration was high. By analyzing the micromechanics and composition at the single aggregate length scale, we identified a strong threshold relationship between local strain and compositions. Namely at the aggrecan concentration below 0.015 arbitrary unit (A.U.) and the collagen concentration above 0.15 A.U., the constructs experienced greater than threefold increase in compressive strain. Overall, this study suggests that local compositional features are the primary driver of the local mechanical environment in tissue-engineered cartilage constructs, providing insight into potential quality control parameters for manufacturing tissue-engineered constructs.

Investigation of the biological activity of hydroxyapatite on vascular endothelial cells

ObjectiveThis study aims to explore the impact of nano-hydroxyapatite (na-HA) and micron-hydroxyapatite (mi-HA) on human umbilical vein endothelial cells (HUVEC) using in vitro experiments, assessing their influence on cellular biological activity. These findings offer crucial experimental data for informing the development of more vascularized tissue-engineered bone constructs. MethodsWe employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of various concentrations of both HA extracts on HUVEC metabolic activity post 48, 72, and 96 h of treatment. Transwell experiments were conducted to evaluate the influence of HA extract on HUVEC migratory capabilities. The cell proliferation activity was assessed using the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, elucidating the impact of varying concentrations of both HA extracts on cell proliferation. Lumen formation experiments were conducted to assess the capacity of HA-treated HUVECs to form lumen-like structures. The Enzyme-Linked Immunosorbent Assay (ELISA) was employed to measure the impact of HA extract on vascular endothelial growth factor (VEGF) secretion by HUVECs. Western blotting (WB) was utilized to analyze alterations in the expression levels of PI3K/Akt signaling pathway-related proteins following HA extract treatment of cells. ResultsAt extract concentrations of 100 g/L and 12.5 g/L, both the mi-HA and na-HA groups demonstrated suppression of cell metabolic activity, migration, and proliferation. Conversely, at 25 g/L, increased cell metabolic activity and proliferative activity were observed. Lumen formation experiments demonstrated that both HA extracts at 100 g/L concentration facilitated lumen formation, with the na-HA group at 25 g/L concentration displaying a more pronounced impact on lumen formation. The ELISA results indicated a notable reduction in VEGF secretion within the mi-HA group at a concentration of 100 g/L. WB experiments revealed that within the na-HA group, treatment of HUVECs with 25 g/L and 12.5 g/L extract concentrations led to upregulation of PI3K and Akt protein expression, while at 100 g/L concentration, Akt protein expression decreased. In the mi-HA group, intracellular expression of both PI3K and Akt proteins exhibited reduction. ConclusionHydroxyapatite extract at both high and low concentrations impacts the biological activity of vascular endothelial cells, with the potential mechanism of action involving the PI3K/Akt signaling pathway.

In vivo biocompatibility testing of nanoparticle-functionalized alginate–chitosan scaffolds for tissue engineering applications

Background: There is a strong interest in designing new scaffolds for their potential application in tissue engineering and regenerative medicine. The incorporation of functionalization molecules can lead to the enhancement of scaffold properties, resulting in variations in scaffold compatibility. Therefore, the efficacy of the therapy could be compromised by the foreign body reaction triggered after implantation.Methods: In this study, the biocompatibilities of three scaffolds made from an alginate–chitosan combination and functionalized with gold nanoparticles (AuNp) and alginate-coated gold nanoparticles (AuNp + Alg) were evaluated in a subcutaneous implantation model in Wistar rats. Scaffolds and surrounding tissue were collected at 4-, 7- and 25-day postimplantation and processed for histological analysis and quantification of the expression of genes involved in angiogenesis, macrophage profile, and proinflammatory (IL-1β and TNFα) and anti-inflammatory (IL-4 and IL-10) cytokines.Results: Histological analysis showed a characteristic foreign body response that resolved 25 days postimplantation. The intensity of the reaction assessed through capsule thickness was similar among groups. Functionalizing the device with AuNp and AuNp + Alg decreased the expression of markers associated with cell death by apoptosis and polymorphonuclear leukocyte recruitment, suggesting increased compatibility with the host tissue. Similarly, the formation of many foreign body giant cells was prevented. Finally, an increased detection of alpha smooth muscle actin was observed, showing the angiogenic properties of the elaborated scaffolds.Conclusion: Our results show that the proposed scaffolds have improved biocompatibility and exhibit promising potential as biomaterials for elaborating tissue engineering constructs.

Engineering large and geometrically controlled vascularized nerve tissue in collagen hydrogels to restore large-sized volumetric muscle loss

Developing scalable vascularized and innervated tissue is a critical challenge for the successful clinical application of tissue-engineered constructs. Collagen hydrogels are extensively utilized in cell-mediated vascular network formation because of their naturally excellent biological properties. However, the substantial increase in hydrogel contraction induced by populated cells limits their long-term use. Previous studies attempted to mitigate this issue by concentrating collagen pre-polymer solutions or synthesizing covalently crosslinked collagen hydrogels. However, these methods only partially reduce hydrogel contraction while hindering blood vessel formation within the hydrogels. To address this challenge, we introduced additional support in the form of a supportive spacer to counteract the contraction forces of populated cells and prevent hydrogel contraction. This approach was found to promote cell spreading, resist hydrogel contraction, control hydrogel/tissue geometry, and even facilitate the engineering of functional blood vessels and host nerve growth in just one week. Subsequently, implanting these engineered tissues into muscle defect sites resulted in timely anastomosis with the host vasculature, leading to enhanced myogenesis, increased muscle innervation, and the restoration of injured muscle functionality. Overall, this innovative strategy expands the applicability of collagen hydrogels in fabricating large vascularized nerve tissue constructs for repairing volumetric muscle loss (∼63 %) and restoring muscle function.

A Dexamethasone-Loaded Polymeric Electrospun Construct as a Tubular Cardiovascular Implant.

Cardiovascular tissue engineering is providing many solutions to cardiovascular diseases. The complex disease demands necessitating tissue-engineered constructs with enhanced functionality. In this study, we are presenting the production of a dexamethasone (DEX)-loaded electrospun tubular polymeric poly(l-lactide) (PLA) or poly(d,l-lactide-co-glycolide) (PLGA) construct which contains iPSC-CMs (induced pluripotent stem cell cardiomyocytes), HUVSMCs (human umbilical vein smooth muscle cells), and HUVECs (human umbilical vein endothelial cells) embedded in fibrin gel. The electrospun tube diameter was calculated, as well as the DEX release for 50 days for 2 different DEX concentrations. Furthermore, we investigated the influence of the polymer composition and concentration on the function of the fibrin gels by imaging and quantification of CD31, alpha-smooth muscle actin (αSMA), collagen I (col I), sarcomeric alpha actinin (SAA), and Connexin 43 (Cx43). We evaluated the cytotoxicity and cell proliferation of HUVECs and HUVSMCs cultivated in PLA and PLGA polymeric sheets. The immunohistochemistry results showed efficient iPSC-CM marker expression, while the HUVEC toxicity was higher than the respective HUVSMC value. In total, our study emphasizes the combination of fibrin gel and electrospinning in a functionalized construct, which includes three cell types and provides useful insights of the DEX release and cytotoxicity in a tissue engineering perspective.