Collagen and Beyond: A Comprehensive Comparison of Human ECM Properties Derived from Various Tissue Sources for Regenerative Medicine Applications.

Collagen, along with proteoglycans, glycosaminoglycans, glycoproteins, and various growth factors, forms the extracellular matrix (ECM) and contributes to the complexity and diversity of different tissues. Herein, we compared the physicochemical and biological properties of ECM hydrogels derived from four different human tissues: skin, bone, fat, and birth. Pure human collagen type I hydrogels were used as control. Physical characterization of ECM hydrogels and assessment of cell response of cord-tissue mesenchymal stem cells (CMSCs) were performed. Decellularization efficiency was found to be >90% for all ECM. Hydroxyproline quantification assay showed that collagen content in birth ECM was comparable to collagen control and significantly greater than other sources of ECM. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of γ, ß, α1 and α2 collagen chains in all ECMs. Gelation kinetics of ECM hydrogels was significantly slower than collagen control. Compressive modulus of skin ECM was the highest and birth ECM was the lowest. Skin and birth ECM hydrogels were more stable than bone and fat ECM hydrogels. CMSCs encapsulated in birth ECM hydrogels exhibited the highest metabolic activity. Rheological characterization revealed that all ECM-derived inks exhibited shear thinning properties, and skin-derived ECM inks were most suitable for extrusion-based bioprinting for the concentration and printing conditions used in this study. Overall, results demonstrate that the physicochemical and biological properties of ECM hydrogels vary significantly depending on the tissue source. Therefore, careful selection of tissue source is important for development of ECM-based biomimetic tissue constructs for regenerative medicine applications.

3D bioprinting of an implantable xeno-free vascularized human skin graft.

Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even autoimmunity. Here, we report successful xeno-free isolation, expansion, and cryopreservation of human endothelial cells (EC), fibroblasts (FBs), pericytes (PCs), and keratinocytes (KCs). We further demonstrate the bioprinting of a human skin substitute with a dermal layer containing xeno-free cultured human EC, FBs, and PCs in a xeno-free bioink containing human collagen type I and fibronectin layered in a biocompatible polyglycolic acid mesh and subsequently seeded with xeno-free human KCs to form an epidermal layer. Following implantation of such bilayered skin grafts on the dorsum of immunodeficient mice, KCs form a mature stratified epidermis with rete ridge-like structures. The ECs and PCs form human EC-lined perfused microvessels within 2 weeks after implantation, preventing graft necrosis, and eliciting further perfusion of the graft by angiogenic host microvessels. As proof-of-concept, we generated 12 individual grafts using a single donor of all four cell types. In summary, we describe the fabrication of a bioprinted vascularized bilayered skin substitute under completely xeno-free culture conditions demonstrating feasibility of a xeno-free approach to complex tissue engineering.

Native human collagen type I provides a viable physiologically relevant alternative to xenogeneic sources for tissue engineering applications: A comparative in vitro and in vivo study.

Xenogeneic sources of collagen type I remain a common choice for regenerative medicine applications due to ease of availability. Human and animal sources have some similarities, but small variations in amino acid composition can influence the physical properties of collagen, cellular response, and tissue remodeling. The goal of this work is to compare human collagen type I-based hydrogels versus animal-derived collagen type I-based hydrogels, generated from commercially available products, for their physico-chemical properties and for tissue engineering and regenerative medicine applications. Specifically, we evaluated whether the native human skin type I collagen could be used in the three most common research applications of this protein: as a substrate for attachment and proliferation of conventional 2D cell culture; as a source of matrix for a 3D cell culture; and as a source of matrix for tissue engineering. Results showed that species and tissue specific variations of collagen sources significantly impact the physical, chemical, and biological properties of collagen hydrogels including gelation kinetics, swelling ratio, collagen fiber morphology, compressive modulus, stability, and metabolic activity of hMSCs. Tumor constructs formulated with human skin collagen showed a differential response to chemotherapy agents compared to rat tail collagen. Human skin collagen performed comparably to rat tail collagen and enabled assembly of perfused human vessels in vivo. Despite differences in collagen manufacturing methods and supplied forms, the results suggest that commercially available human collagen can be used in lieu of xenogeneic sources to create functional scaffolds, but not all sources of human collagen behave similarly. These factors must be considered in the development of 3D tissues for drug screening and regenerative medicine applications.

In vitro characterization of xeno-free clinically relevant human collagen and its applicability in cell-laden 3D bioprinting.

Collagen type I, commonly derived from xenogenic sources, is extensively used as a biomaterial for tissue engineering applications. However, the use of xenogenic collagen is typically associated with species specific variation in mechanical, structural, and biological properties that are known to influence cellular response and remodeling. In addition, immunological complications and risks of disease transmission are also major concerns. The goal of this study is to characterize a new xeno-free human skin-derived collagen and assess its applicability as a bioink for cell-laden 3 D bioprinting. Four different concentrations of human collagen (i.e., 0.5 mg/mL, 1 mg/mL, 3 mg/mL and 6 mg/mL) were employed for the synthesis of collagen hydrogels. In addition, bovine collagen was used as a xenogenic control. Results from SDS-PAGE analysis showed the presence of α1, α2, and ß chains, confirming that the integrity of type I human collagen is maintained post isolation. Polymerization rate and compressive modulus increased significantly with increase in the concentration of human collagen. When comparing two different sources of collagen, the polymerization rate of xenogenic collagen was significantly faster (p < 0.05) than human collagen while the compressive modulus was comparable. Raman spectroscopy showed a large peak in the Amide I band around 1600 cm-1, indicating a dense and supraorganized fibrillar structure in human collagen hydrogels. Conversely, Amide I band intensity for xenogenic collagen was comparable to that of Amide II and Amide III bands. Further, the use of 6 mg/mL human collagen as a bioink yielded 3 D printed constructs with high shape fidelity and cell viability. On the other hand, xenogenic collagen failed to yield stable 3 D printed constructs. Together, the results from this study provides an impetus for using human-derived collagen as a viable alternative to xenogenic sources for 3 D bioprinting of clinically relevant scaffolds for tissue engineering applications.

Regen med therapeutic opportunities for fighting COVID-19.

This perspective from a Regenerative Medicine Manufacturing Society working group highlights regenerative medicine therapeutic opportunities for fighting COVID-19. This article addresses why SARS-CoV-2 is so different from other viruses and how regenerative medicine is poised to deliver new therapeutic opportunities to battle COVID-19. We describe animal models that depict the mechanism of action for COVID-19 and that may help identify new treatments. Additionally, organoid platforms that can recapitulate some of the physiological properties of human organ systems, such as the lungs and the heart, are discussed as potential platforms that may prove useful in rapidly screening new drugs and identifying at-risk patients. This article critically evaluates some of the promising regenerative medicine-based therapies for treating COVID-19 and presents some of the collective technologies and resources that the scientific community currently has available to confront this pandemic.

3D printed mitral valve models: affordable simulation for robotic mitral valve repair.

OBJECTIVES: 3D printed mitral valve (MV) models that capture the suture response of real tissue may be utilized as surgical training tools. Leveraging clinical imaging modalities, 3D computerized modelling and 3D printing technology to produce affordable models complements currently available virtual simulators and paves the way for patient- and pathology-specific preoperative rehearsal. METHODS: We used polyvinyl alcohol, a dissolvable thermoplastic, to 3D print moulds that were casted with liquid platinum-cure silicone yielding flexible, low-cost MV models capable of simulating valvular tissue. Silicone-moulded MV models were fabricated for 2 morphologies: the normal MV and the P2 flail. The moulded valves were plication and suture tested in a laparoscopic trainer box with a da Vinci Si robotic surgical system. One cardiothoracic surgery fellow and 1 attending surgeon qualitatively evaluated the ability of the valves to recapitulate tissue feel through surveys utilizing the 5-point Likert-type scale to grade impressions of the valves. RESULTS: Valves produced with the moulding and casting method maintained anatomical dimensions within 3% of directly 3D printed acrylonitrile butadiene styrene controls for both morphologies. Likert-type scale mean scores corresponded with a realistic material response to sutures (5.0/5), tensile strength that is similar to real MV tissue (5.0/5) and anatomical appearance resembling real MVs (5.0/5), indicating that evaluators 'agreed' that these aspects of the model were appropriate for training. Evaluators 'somewhat agreed' that the overall model durability was appropriate for training (4.0/5) due to the mounting design. Qualitative differences in repair quality were notable between fellow and attending surgeon. CONCLUSIONS: 3D computer-aided design, 3D printing and fabrication techniques can be applied to fabricate affordable, high-quality educational models for technical training that are capable of differentiating proficiency levels among users.

Validation and cultural adaptation of the Arabic versions of the Mini-Mental Status Examination - 2 and Mini-Cog test.

INTRODUCTION: The elderly population is increasing around the world, and the prevalence of dementia increases with age. Hence, it is expected that the number of people with dementia will increase significantly in the coming years. The Mini-Mental Status Examination - 2 (MMSE-2) and Mini-Cog are widely used tests to screen for dementia. These scales have good reliability and validity and are easy to administer in clinical and research settings. AIM: The purpose of this study was to validate the Arabic versions of MMSE-2 and Mini-Cog. These scales were assessed against the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria for dementia, as the gold standard. METHODS: The standard versions of the MMSE-2 and Mini-Cog were translated to Arabic following the back-translation method. Then, a trained rater administered these tests to 134 Arab elderly aged >60 years. A physician, blind to the results of these two tests, assessed the participants for vascular dementia or probable Alzheimer's disease, based on the DSM-IV-TR criteria. RESULTS: The sample included 67.2% Qataris. The mean age was 74.86 years (standard deviation =7.71), and 61.9% did not attend school. The mean of the adjusted scores of MMSE-2 based on age and education level was 19.60 (standard deviation =6.58). According to DSM-IV-TR, 17.2% of the participants had dementia. Sensitivity and specificity of the MMSE-2 and the Mini-Cog together were 71.4% and 61.6%, respectively, which were better than those of each test alone. CONCLUSION: Together, the Arabic versions of MMSE-2 and Mini-Cog are good screening tools for cognitive impairment in Arabs.

Common and Rare Genetic Variants Associated With Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most devastating disorders. Despite the continuing increase of its incidence among aging populations, no effective cure has been developed mainly due to difficulties in early diagnosis of the disease before damaging of the brain, and the failure to explore its complex underlying molecular mechanisms. Recent technological advances in genome-wide association studies (GWAS) and high throughput next generation whole genome, and exome sequencing had deciphered many of AD-related loci, and discovered single nucleotide polymorphisms (SNPs) that are associated with altered AD molecular pathways. Highlighting altered molecular pathways linked to AD pathogenesis is crucial to identify novel diagnostic and therapeutic AD targets.

Chitosan fibers with improved biological and mechanical properties for tissue engineering applications.

The low mechanical properties of hydrogel materials such as chitosan hinder their broad utility for tissue engineering applications. Previous research efforts improved the mechanical properties of chitosan fiber through chemical and physical modifications; however, unfavorable toxicity effects on cells were reported. In this paper, we report the preparation of chitosan fibers with improved mechanical and biocompatibility properties. The structure-property relationships of extruded chitosan fibers were explored by varying acetic acid (AA) concentration, ammonia concentration, annealing temperature and degree of heparin crosslinking. Results showed that optimizing AA concentration to 2vol% improved fiber strength and stiffness by 2-fold. Extruding chitosan solution into 25wt% of ammonia solution reduced fiber diameters and improved fiber strength by 2-fold and stiffness by 3-fold, due to an increase in crystallinity as confirmed by XRD. Fiber annealing further reduced fiber diameter and improved fiber strength and stiffness as temperature increased. Chitosan fibers crosslinked with heparin had increased diameter but lower strength and stiffness properties and higher breaking strain values. When individual parameters were combined, further improvement in fiber mechanical properties was achieved. All mechanically improved fibers and heparin crosslinked fibers promoted valvular interstitial cells (VIC) attachment and growth over 10 day cultures. Our results demonstrate the ability to substantially improve the mechanical properties of chitosan fibers without adversely affecting their biological properties. The investigated treatments offer numerous advantages over previous physical/chemical modifications and thus are expected to expand the utility of chitosan fibers with tunable mechanical properties in various tissue engineering applications.

Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology.

This study was designed to develop a versatile method for fabricating complex and heterogeneous three-dimensional (3D) tissue constructs using simultaneous ink-jetting of multiple cell types. Human amniotic fluid-derived stem cells (hAFSCs), canine smooth muscle cells (dSMCs), and bovine aortic endothelial cells (bECs), were separately mixed with ionic cross-linker calcium chloride (CaCl(2)), loaded into separate ink cartridges and printed using a modified thermal inkjet printer. The three cell types were delivered layer-by-layer to pre-determined locations in a sodium alginate-collagen composite located in a chamber under the printer. The reaction between CaCl(2) and sodium alginate resulted in a rapid formation of a solid composite gel and the printed cells were anchored in designated areas within the gel. The printing process was repeated for several cycles leading to a complex 3D multi-cell hybrid construct. The biological functions of the 3D printed constructs were evaluated in vitro and in vivo. Each of the printed cell types maintained their viability and normal proliferation rates, phenotypic expression, and physiological functions within the heterogeneous constructs. The bioprinted constructs were able to survive and mature into functional tissues with adequate vascularization in vivo. These findings demonstrate the feasibility of fabricating complex heterogeneous tissue constructs containing multiple cell types using inkjet printing technology.

Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications.

Bioprinting is an emerging technique used to fabricate viable, 3D tissue constructs through the precise deposition of cells and hydrogels in a layer-by-layer fashion. Despite the ability to mimic the native properties of tissue, printed 3D constructs that are composed of naturally-derived biomaterials still lack structural integrity and adequate mechanical properties for use in vivo, thus limiting their development for use in load-bearing tissue engineering applications, such as cartilage. Fabrication of viable constructs using a novel multi-head deposition system provides the ability to combine synthetic polymers, which have higher mechanical strength than natural materials, with the favorable environment for cell growth provided by traditional naturally-derived hydrogels. However, the complexity and high cost associated with constructing the required robotic system hamper the widespread application of this approach. Moreover, the scaffolds fabricated by these robotic systems often lack flexibility, which further restrict their applications. To address these limitations, advanced fabrication techniques are necessary to generate complex constructs with controlled architectures and adequate mechanical properties. In this study, we describe the construction of a hybrid inkjet printing/electrospinning system that can be used to fabricate viable tissues for cartilage tissue engineering applications. Electrospinning of polycaprolactone fibers was alternated with inkjet printing of rabbit elastic chondrocytes suspended in a fibrin-collagen hydrogel in order to fabricate a five-layer tissue construct of 1 mm thickness. The chondrocytes survived within the printed hybrid construct with more than 80% viability one week after printing. In addition, the cells proliferated and maintained their basic biological properties within the printed layered constructs. Furthermore, the fabricated constructs formed cartilage-like tissues both in vitro and in vivo as evidenced by the deposition of type II collagen and glycosaminoglycans. Moreover, the printed hybrid scaffolds demonstrated enhanced mechanical properties compared to printed alginate or fibrin-collagen gels alone. This study demonstrates the feasibility of constructing a hybrid inkjet printing system using off-the-shelf components to produce cartilage constructs with improved biological and mechanical properties.

Improving the mechanical properties of chitosan-based heart valve scaffolds using chitosan fibers.

Chitosan is being widely studied for tissue engineering applications due to its biocompatibility and biodegradability. However, its use in load-bearing applications is limited due to low mechanical properties. In this study, we investigated the effectiveness of a chitosan fiber reinforcement approach to enhancing the mechanical properties of chitosan scaffolds. Chitosan fibers were fabricated using a solution extrusion and neutralization method and incorporated into porous chitosan scaffolds. The effects of fiber/scaffold mass ratio, fiber mechanical properties and fiber length on scaffold mechanical properties were studied. The results showed that incorporating fibers improved scaffold strength and stiffness in proportion to the fiber/scaffold mass ratio. A fiber-reinforced, heart valve scaffold achieved leaflet tensile strength values of 220±17 kPa, comparable to the radial values of human pulmonary valve leaflets. Additionally, the effects of 2 mm fibers were found to be up to threefold greater than 10 mm fibers at identical mass ratios. Heparin crosslinking of fibers produced a reduction in fiber strength, and thus failed to produce additional improvements to fiber-reinforced scaffold properties. Despite this reduction in fiber strength, heparin-modified fibers still improved the mechanical properties of reinforced scaffolds, but to a lesser extent than unmodified fibers. The results demonstrate that chitosan fiber reinforcement can be used to achieve porous chitosan scaffold strength approaching that of tissue, and that fiber length and mechanical properties are important parameters in defining the degree of mechanical improvement.

Membrane thickness is an important variable in membrane scaffolds: Influence of chitosan membrane structure on the behavior of cells.

Cell and tissue responses to polymeric materials are orchestrated in part by the conformations of adsorbed plasma proteins. Thus, the chemical properties of a polymer membrane that govern protein adsorption behavior can play an important role in determining the biological properties of tissue engineered scaffolds derived from that polymer. In this study, we explored the role of membrane thickness as a factor influencing cell adhesion and proliferation on chitosan membranes with and without covalently attached glycosaminoglycans. Rat mesenchymal stem cells (MSCs) cultured on chitosan membranes of various thicknesses demonstrated significantly improved cell adhesion, spreading and proliferation as membrane thickness was increased. Rat hepatocytes displayed increased spreading on the substrate with increasing membrane thickness, similar to MSCs. Increased thickness reduced the overall crystallinity of the membrane, and the data indicate that the improved cellular responses were likely due to enhanced adsorption of serum vitronectin, presumably due to reduced membrane crystallinity. These results demonstrate that membrane thickness is an important design variable that can be manipulated in chitosan-based scaffolds to achieve enhanced cell spreading, proliferation and function.

Does the use of erythropoietin reduce the risk of exposure to allogeneic blood transfusion in cardiac surgery? A systematic review and meta-analysis.

BACKGROUND: The use of blood conservation techniques is important in cardiac surgery as postoperative bleeding is common and allogeneic blood transfusion carries the risk of transfusion reactions and infection transmission. Erythropoietin with and without preoperative autologous blood donation is one of the modalities to avoid allogeneic blood transfusion. The objective of this review was to assess the effectiveness of erythropoietin in reducing the risk of exposure to allogeneic blood transfusion during or after cardiac surgery. METHODS: A meta-analysis of 11 identified randomized controlled trials, reporting comparisons between erythropoietin and control, was undertaken. The primary outcome was the number of patients exposed to allogeneic blood transfusion during or after cardiac surgery. RESULTS: Eleven studies, involving 708 patients, met the inclusion criteria for this review. In total, 471 patients were given erythropoietin, and 237 patients formed the control group. The administration of erythropoietin with and without preoperative autologous blood transfusion prior to cardiac surgery is associated with a significant risk reduction: RR = 0.28 (95% CI 0.18-0.44, P < 0.001) and RR = 0.53 (95% CI 0.32-0.88, P < 0.01), respectively. CONCLUSION: The administration of erythropoietin before cardiac surgery is associated with a significant reduction in the risk of exposure to allogeneic blood transfusion. Further studies are warranted to define the patients' subgroups that may benefit the most from EPO administration.