Autologous Tissue Engineering Research Articles

Modification of plasma protein for bioprinting via photopolymerization

Bioprinting is emerging as an advanced tool in tissue engineering. However, there is still a lack of bioinks able to form hydrogels with desirable bioactivities that support positive cell behaviors. In this study, modified plasma proteins capable of forming hydrogels with multiple biological functions are developed as bioinks for digital light processing (DLP) printing. The Plasma-MA (BM) was synthesized via a one-pot method through the reaction between the fresh frozen plasma and methacrylic anhydride. The methacrylated levels were observed to influence the physical properties of BM hydrogels including mechanical properties, swelling, and degradation. The photo-crosslinked BM hydrogels can sustainedly release vascular endothelial growth factor (VEGF) and exhibit positive biological effects on cell adhesion and proliferation, and cell functionality such as tube formation of human umbilical vein endothelial cells (HUVECs), and neurite elongation of rat pheochromocytoma cells (PC12). Meanwhile, BM hydrogels can also induce cell infiltration, modulate immune response, and promote angiogenesis in vivo. Moreover, the plasma bioinks can be used to fabricate customized scaffolds with complex structures through a DLP printing process. These findings implicate that the modified plasma with growth factor release is a promising candidate for bioprinting in autologous and personalized tissue engineering.

Engineered exosomes reprogram Gli1+ cells in vivo to prevent calcification of vascular grafts and autologous pathological vessels.

Calcification of autologous pathological vessels and tissue engineering blood vessels (TEBVs) is a thorny problem in clinic. However, there is no effective and noninvasive treatment that is available against the calcification of TEBVs and autologous pathological vessels. Gli1+ cells are progenitors of smooth muscle cells (SMCs) and can differentiate into osteoblast-like cells, leading to vascular calcification. Our results showed that the spatiotemporal distribution of Gli1+ cells in TEBVs was positively correlated with the degree of TEBV calcification. An anticalcification approach was designed consisting of exosomes derived from mesenchymal stem cells delivering lncRNA-ANCR to construct the engineered exosome-Ancr/E7-EXO. The results showed that Ancr/E7-EXO effectively targeted Gli1+ cells, promoting rapid SMC reconstruction and markedly inhibiting Gli1+ cell differentiation into osteoblast-like cells. Moreover, Ancr/E7-EXO significantly inhibited vascular calcification caused by chronic kidney disease. Therefore, Ancr/E7-EXO reprogrammed Gli1+ cells to prevent calcification of vascular graft and autologous pathological vessel, providing unique insights for an effective anticalcification.

Improved contractile potential in detrusor microtissues from pediatric patients with end stage lower urinary tract dysfunction.

Autologous cell-based tissue engineering has been proposed as a treatment option for end stage lower urinary tract dysfunction (ESLUTD). However, it is generally accepted that cells isolated from patient bladders retain the pathological properties of their tissue of origin and therefore need to be improved before they can serve as a cell source for tissue engineering applications. We hypothesize that human three-dimensional (3D) microtissues of detrusor smooth muscle cells (SMCs) are valuable ex vivo disease models and potent building blocks for bladder tissue engineering. Detrusor SMCs isolated from bladder wall biopsies of pediatric ESLUTD patients and healthy controls were expanded and cultured into 3D microtissues. Gene and protein analyses were performed to explore the effect of microtissue formation on SMC viability, contractile potential, bladder wall specific extracellular matrix (ECM) composition and mediators of ECM remodeling. Through microtissue formation, remodeling and intensified cell-cell interactions, the ESLUTD SMCs lost their characteristic disease phenotype. These microtissues exhibited similar patterns of smooth muscle related contractile proteins and essential bladder wall-specific ECM components as microtissues from healthy control subjects. Thus, the presented data suggest improved contractile potential and ECM composition in detrusor SMC microtissues from pediatric ESLUTD patients. These findings are of great relevance, as 3D detrusor SMC microtissues might be an appropriate cell source for autologous cell-based bladder tissue engineering.

Feeder-Free Cell Culture of Labial Oral Mucosal Epithelium for Tissue-Engineering and Regenerative Medicine

Background.The cultured cheek mucosa epithelium (buccal epithelium, BE) is used for autologous transplants generation and tissue engineering. An alternative source of cells for these purposes may be the lip mucosa, covered, like BE, with non-keratinized stratified squamous epithelium, but with some histological distinctions.Aims to characterize the human lip mucosa as a promising source of epithelial cells for autologous transplantation and tissue engineering.Methods.Scrapings of the lip, cheek, and gum mucosa from five healthy volunteers were analyzed by cytofluorimetry to determine the level of desquamation and cytokeratin (CK) 10 and 13 expression. The lip mucosa of two patients was characterized using routine histological staining and fluorescence immunohistochemistry for CK 3, 4, 10, 13, and p63 marker. 35 samples of full-thickness strips of the patients lip mucosa were used to set the explant (n=18) and enzymatic (n=17) techniques for expansion epithelium. Culture systems with 1.05 and 0.06mM Calcium contained 5% fetal bovine serum, 5 g/ml human insulin, 5 g/ml hydrocortisone, 10 ng/ml human epidermal growth factor. Stable cultures were stained for p63, vimentin, zonula occludens-1 (ZO-1), and CK10. Software tools determined levels of their expression.Results.The number of cells in the lip and gum samples was significantly lower than from the cheek. The median number of CK13 positive cells was significantly different for the gum (6.4%) and cheek (64.8%, p=0.0089). Significant differences for CK10 positive cells were not observed. The epithelium of the lip mucosa was 72.13.6 m thick, relatively flat, and without keratinization sites. Samples were positively stained for CK 4 and 13, in the absence of expression of CK 3 and 10. The primary culture of epithelial cells obtained by explant technique was significantly more effective (p=0.001) in comparison with the enzymatic method. Stable cultures had a cobble-stone morphology in both culture systems. The levels of vimentin and p63 expression in both culture systems was not significantly differ. ZO-1 expression was 3.6-fold higher for 1.05-mMCa++medium (p=0.0006).Conclusions.Epithelium cell culture from the lip mucosa can be obtained by culturing explants without a feeder layer. Quality control steps have been developed for cultured cells and biopsy site.

Autologous protein-based scaffold composed of platelet lysate and aminated hyaluronic acid.

This study describes a protein-based scaffold using platelet rich plasma (PRP), aminated hyaluronic acid (HA-NH2) and Genipin for potential use in regenerative applications as an autologous tissue engineering scaffold. Human PRP was subjected to three freeze-thaw cycles for obtaining platelet lysates (PL). HA-NH2 was synthesized from hyaluronic acid. PL/HA-NH2 scaffolds were fabricated using different concentrations of genipin (0.05, 0.1 and 0.2%) and HA-NH2 (10, 20 and 30 mg/mL). Mechanical, physical, and chemical properties of the scaffolds were comprehensively investigated. The compressive test findings revealed that crosslinking with 0.1 and 0.2% genipin improved the mechanical properties of the scaffolds. SEM evaluations showed that the scaffolds exhibited an interconnected and macroporous structure. Besides, porosimetry analysis indicated a wide distribution of the scaffold pore-size. Rheological findings demonstrated that the G' values were higher than the G″ values, indicating that PL/HA-NH2 scaffolds had typical viscoelastic properties. In vitro biocompatibility studies showed that the scaffolds were both cytocompatible and hemocompatible. Alamar Blue test indicated that human adipose mesenchymal stem cells (hASCs) were able to attach, spread and proliferate on the scaffolds for 21 days-duration. Our findings clearly indicate that PL/HA-NH2 can be a promising autologous candidate scaffold for tissue engineering applications.

AB009. Bioprosthetics in airway surgery

: Among many challenges in thoracic surgery, tracheal substitutes have been particularly elusive over nearly 75 years. Although nearly 4–5 cm of trachea can be resected and primarily reconstructed in properly selected patients, larger or more complicated resections have spawned innovation to overcome this limitation. The trachea functions as an airtight, mechanically stable passage from larynx to bronchi and facilitates clearance of secretions via a specialized epithelium. Ideal properties of a tracheal substitute include durable, non-immunogenic material that is resistant to infection and can easily incorporate surrounding tissue. Four methodologies have been implemented for airway reconstruction: autologous tissue, allotransplantation, bioprosthetics, and tissue engineering. Of note, complete circumferential prosthetic tracheal substitutes have historically failed. The most innovative solutions to tracheal replacement have been tissue engineering and bioprosthetics but both strategies have significant limitations. Tubularized aortic homograft supported by an endoluminal internal stent (Montgomery tube, silicone T-tube or nitinol stent) and external buttressing with muscle has been studied in patients with some limited success. A hypothesis has been put forward from some authors that aortic homografts contain undifferentiated stem cells that may play a role in migration, proliferation and cell signaling of host cells to form primitive tracheal elements including intima replacement with squamous or ciliated epithelium and chondrogenesis. In a few cases the internal stents were removed beyond 1–2 years with regeneration of cartilage elements within the graft. Despite this interesting biology, reported long-term complications include infection and tracheoesophageal fistula. Both aortic homograft and human acellular dermal matrix have experienced more success when implemented as a patch repair of large non-circumferential airway defects. The etiology of these defects can be derived from trauma with loss of tissue domain, infection and erosion (such as tracheoesophageal fistulae), or partial resection for neoplasm. In the case of human acellular dermal matrix the bioprosthetic is meticulously sutured so that the dermal side faces the airway lumen. Since this tissue matrix normally is broken down by the host, it primary serves as a scaffold for tissue ingrowth. Of note, the luminal side of aortic homograft is also oriented to face the airway lumen and the wall is perforated with a 16-gauge needle to promote tissue ingrowth and neovascularization. In the description of these reconstructions, repairs were routinely buttressed with vascularized tissue exteriorly and granulation tissue was observed at the site of reconstruction in many patients with mild tissue contracture and eventual native tissue ingrowth. It is understood that these patients require frequent surveillance bronchoscopy with debridement of granulation as necessary. Reported complications of this strategy were sputum retention, pneumonia and pneumomediastinum. One author reports selective use of hyperbaric oxygen in patients with complex repairs to enhance tissue ingrowth of the bioprosthetic. Tissue engineering generally involves the implantation of a biodegradable scaffold seeded with host cells that can repopulate the scaffold with new extracellular matrix. This has been reported in experimental settings with decellularized cadaveric trachea seeded with bone-marrow derived mesenchymal stroma cells as well as autologous respiratory epithelium. Initial attempts at tracheal tissue engineering showed promise in preclinical models but were associated with unacceptable outcomes in humans. Lastly, several studies have been published implementing three-dimensional (3D) printing technology to engineer tracheal substitutes that mimic mechanical behavior similar to native trachea. Biodegradable scaffolds of trachea have been 3D printed, some cultured with chondrocytes allowing formation of neocartilage, and tested in preclinical models. Although these investigations are very preliminary, 3D printing appears to provide infinite possibilities for tissue engineering.

Extracellular matrix derived by human umbilical cord-deposited mesenchymal stem cells accelerates chondrocyte proliferation and differentiation potential in vitro.

The extracellular matrix (ECM) is a dynamic and intricate three-dimensional (3D) microenvironment with excellent biophysical, biomechanical, and biochemical properties that may directly or indirectly regulate cell behavior, including proliferation, adhesion, migration, and differentiation. Compared with tissue-derived ECM, cell-derived ECM potentially has more advantages, including less potential for pathogen transfer, fewer inflammatory or anti-host immune responses, and a closer resemblance to the native ECM microenvironment. Different types of cell-derived ECM, such as adipose stem cells, synovium-derived stem cells and bone marrow stromal cells, their effects on articular chondrocytes which have been researched. In this study, we aimed to develop a 3D cell culture substrate using decellularized ECM derived from human umbilical cord-derived mesenchymal stem cells (hUCMSCs), and evaluated the effects on articular chondrocytes. We evaluated the morphology and components of hUCMSC-derived ECM using physical and chemical methods. Morphological, histological, immunohistochemical, biochemical, and real-time PCR analyses demonstrated that proliferation and differentiation capacity of chondrocytes using the 3D hUCMSC-derived ECM culture substrate was superior to that using non-coated two-dimensional plastic culture plates. In conclusion, 3D decellularized ECM derived from hUCMSCs offers a tissue-specific microenvironment for in vitro culture of chondrocytes, which not only markedly promoted chondrocyte proliferation but also preserved the differentiation capacity of chondrocytes. Therefore, our findings suggest that a 3D cell-derived ECM microenvironment represents a promising prospect for autologous chondrocyte-based cartilage tissue engineering and regeneration. The hUCMSC-derived ECM as a biomaterial is used for the preparation of scaffold or hybrid scaffold products which need to further study in the future.

DOZ047.67: Comparison of mesenchymal stem cells from pediatric normal and esophageal atresia patients for use in regenerative medicine

Abstract Esophageal atresia (EA) is a congenital defect in which the esophagus is not fully connected. Current treatments to bring together long gaps between the esophagus have very high complication rates and the replacement tissue does not possess the correct structure or physiologic properties. Tissue engineering and regenerative medicine have been suggested as a new patient-specific treatment for EA utilizing the patient's own cells and a tubular scaffold. Our group recently published on the successful regeneration of an esophagus using a retrievable scaffold seeded with adipose derived mesenchymal stem cells (AD-MSC) in a porcine model of esophageal loss. The goal of this study is to evaluate phenotypic differences between AD-MSCs from normal (N-AD-MSC) and EA patients (EA-AD-MSC) to determine if EA-AD-MSC could be used for autologous tissue engineering approaches. Subcutaneous fat was obtained from pediatric patients undergoing surgery (IRB#). The cells from all patients expressed mesenchymal stem cell markers CD90, CD105, CD73, CD44 and did not express hematopoietic markers CD45, CD34 and HLA-DR by flow cytometry. Growth kinetics revealed similar population doubling times and morphology was similar between the two groups. Some of the EA-AD-MSC demonstrated upregulated expression of ATNX1, BMI1, FOXF1, GLI2, GLI3, MYC, HOXC4 and NOG, known genotypic abnormalities in EA patients. None of the N-AD-MSCs had upregulated expression of these genes. Furthermore N-AD-MSCs and EA-AD-MSCs underwent osteogenic and adipogenic differentiation with positive staining for Oil Red O and Alizarian Red after 2 weeks in culture. The integrin profiles of these cells were also similar across all patients as demonstrated by flow cytometry. In conclusion, in vitro, the EA-AD-MSCs behaved similarly to N-AD-MSCs in growth kinetics, mesenchymal phenotype and differentiation ability. Genotypically there were some differences and how this will impact the growth of these cells on scaffolds is currently being investigated.

Hypoxic changes to the urothelium as a bystander of end-stage bladder disease

Urothelial cells harvested from benign diseased bladders have a compromised capacity to propagate or differentiate invitro, potentially limiting their application in autologous tissue engineering approaches. The causative pathways behind this altered phenotype are unknown. The hypothesis is that hypoxic damage to the urothelium occurs as a bystander to chronic or recurrent episodes of infection and inflammation. The aim of this study was to assess immunohistochemically detected nuclear hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor in the urothelium when exposed to hypoxia. Human bladder sections from a total of 29 adult and paediatric patients, representing a variety of different pathologies including neuropathy (n=15), were analysed. Tissues from adults with bladder outlet obstruction secondary to prostatic disease (n=1), urothelial carcinoma (n=1) and tonsil (n=1) were used as positive tissue controls for immunohistochemistry. Hypoxia-inducible factor 1 alpha-labelled sections were scanned using a Zeiss AxioScan Z1 slide scanner. Analysis of urothelial nuclear HIF-1α labelling was performed using HistoQuest image analysis software (TissueGnostics). Comparison of nuclear HIF-1α labelling between neuropathic and non-neuropathic sections was performed using one-way analysis of variance with the post hoc Tukey honestly significant difference (HSD) test. Patient urodynamic studies performed before tissue sample harvest were evaluated and correlated to the HIF-1α intensity using Spearman's rank correlation. Hypoxia-inducible factor 1 alpha appeared more intense in the urothelial compartment from neuropathic bladder samples (n=15) than in the control tissues, including non-obstructedsamples (n=9). Image analysis supported this; median nuclear HIF-1α labelling was 29.98±3.10 (standard deviation [SD]) (n=9) in controls and 74.29±7.55 (SD) in neuropathic samples (n=15). A statistically significant difference between the control and neuropathic tissue groups was shown (P<0.05). Of the 15 neuropathic samples, 11 had traceable urodynamic studies. Both initial and maximum detrusor pressures indicated a positive relationship when plotted against HIF-1α labelling. Spearman's rank correlation, with no missing events, confirmed significant correlations between both initial or maximum detrusor pressure and nuclear HIF-1α labelling intensity (median score); P≤0.046 and P≤0.05, respectively. The null hypothesis was accordingly rejected. This study indicates that urothelial nuclear HIF-1α may be a biomarker of hypoxia and a common feature in end-stage bladder disease associated with high-pressure systems.

The Future of Tissue Engineering in Heart Transplantation.

As our knowledge of biologic therapies used in cardiovascular regenerative medicine grows, our focus is turning to developing a bioartificial heart that can alleviate the shortage of donor hearts. Using knowledge gained during 5 decades' worth of work on mechanical total artificial hearts (TAHs), we are developing tissue-engineered hearts for transplantation.The field of cardiovascular regenerative medicine began in the 1990s, when the first attempts were made to treat advanced heart disease with the use of genes or cells. Since then, biologic therapies have shown promise in slowing the progression of cardiovascular disease, so their use is gradually being accepted; however, they are unlikely to reverse end-stage organ failure. Heart transplantation remains the definitive treatment for the failing heart.As early as the 1960s, when the heart was perceived only as a pump, mechanical devices were being designed to replace a failing organ. The need to build an efficient, functional replacement for transplantable organs has become urgent because clinicians are able to prolong the lives of patients who have acute myocardial infarctions or chronic heart failure, but they cannot cure them. Therefore, an engineered bioartificial heart—a living organ manufactured for transplantation—is viewed as a viable way to overcome shortages of donor organs.Many insights from mechanical TAH development can be applied to engineering a bioartificial heart. Appropriate sizing, interdependency of the organ's left and right sides, antithrombogenicity, and durability are important design criteria for any transplantable heart. However, the use of cells as an energy source, the need for vascularization, and the capability for endogenous repair are unique to a bioartificial organ. Another essential component is a sterile bioreactor system that can feed and support the organ as it matures in a sterile environment at controlled temperatures and atmospheric pressures.A normal, healthy heart comprises specialized cells (cardiomyocytes, endothelium, fibroblasts, neurons, and so on) that are integrated into a scaffold of extracellular matrix (ECM) (Fig. 1), richly infiltrated by a dense network of cardiac vessels.1 Much research has focused on finding cell sources and a suitable vascularized scaffold. Stem cells, initially envisioned for cardiac repair, are a natural source for this milieu. As stem cell biology has progressed, so have methods of growing stem cells, which are being used in vitro to populate cardiac patches and even whole organs. Now that inducible pluripotent stem cell technology has been introduced, autologous tissue engineering is imaginable. However, hearts need hundreds of billions of cardiomyocytes and other cells, and generating that number of cells is a hurdle.Of course, cells alone do not constitute an organ. During development, cells coalesce with ECM in a given orientation to form functional units that are unique to each organ. From our perspective, bioengineering an organ involves recombining cells with ECM of an adult organ from which cells have been removed. This decellularized ECM provides appropriate architectural and biological cues to support form and function.2 It retains organ complexity and structure, including fiber orientation and macro- and microarchitecture, as well as vascular channels to deliver nutrients,3,4 thus enabling full-thickness tissue generation. Furthermore, this ECM contains biologically active molecules that support cell migration, proliferation, and differentiation. Finally, the dynamic reciprocity between cells and the ECM enables both to evolve as functional needs change.To overcome the challenges of generating enough cells to build a whole heart that must then remain sterile in vitro, we are exploring in vivo organ engineering involving decellularized heart scaffolds (with or without re-endothelialized vessels) and the body's capacity to repopulate the scaffolds with cells. Specifically, we have implanted decellularized porcine hearts into living pigs in a heterotopic position. We have found that the surgical circuit created, generated a pressure gradient for, and promoted flow crucial to prevent coagulation and increase graft patency. In addition, short-term implantation promoted endothelial cell adhesion to the vessel lumina, and long-term implantation promoted tissue formation with evidence of cardiomyocytes and recellularized vessels within the graft.5 By adding load and electrical input, possibly with use of temporary mechanical assistance or a pacemaker, we think that vascular and parenchymal cell maturation can occur.We hope that these and other efforts will enable tissue engineering to contribute to heart transplantation in the future.

Decision Support Tools for Regenerative Medicine: Systematic Review.

BackgroundDecisional tools have demonstrated their importance in informing manufacturing and commercial decisions in the monoclonal antibody domain. Recent approved therapies in regenerative medicine have shown great clinical benefits to patients.ObjectiveThe objective of this review was to investigate what decisional tools are available and what issues and gaps have been raised for their use in regenerative medicine.MethodsWe systematically searched MEDLINE to identify articles on decision support tools relevant to tissue engineering, and cell and gene therapy, with the aim of identifying gaps for future decisional tool development. We included published studies in English including a description of decisional tools in regenerative medicines. We extracted data using a predesigned Excel table and assessed the data both quantitatively and qualitatively.ResultsWe identified 9 articles addressing key decisions in manufacturing and product development challenges in cell therapies. The decision objectives, parameters, assumptions, and solution methods were analyzed in detail. We found that all decisional tools focused on cell therapies, and 6 of the 9 reviews focused on allogeneic cell therapy products. We identified no available tools on tissue-engineering and gene therapy products. These studies addressed key decisions in manufacturing and product development challenges in cell therapies, such as choice of technology, through modeling.ConclusionsOur review identified a limited number of decisional tools. While the monoclonal antibodies and biologics decisional tool domain has been well developed and has shown great importance in driving more cost-effective manufacturing processes and better investment decisions, there is a lot to be learned in the regenerative medicine domain. There is ample space for expansion, especially with regard to autologous cell therapies, tissue engineering, and gene therapies. To consider the problem more comprehensively, the full needle-to-needle process should be modeled and evaluated.

Comparison of the characteristics of mesenchymal stem-like cells derived by integration-free induced pluripotent stem cells in different single-cell culture media under feeder-free conditions.

Generating mesenchymal stem-like cells (MSLCs) from induced pluripotent stem cells (iPSCs) can be a practical method for obtaining the sufficient cells for autologous tissue engineering. Single-cell culturing in specific medium and non-feeder cells is an alternative and promising strategy to overcome problems of embryo culture; however, little is known about how different culture media affect the proliferation and differentiation of MSLCs. We first derived MSLCs from iPSCs with non-integrating episomal plasmid vectors (hereafter 409B2 cells) using three different cell culture media, including single-cell culture medium in feeder-free condition: mTeSR1, DEF-CS500, or StemFit AK02N. The morphology of all MSLCs was completely altered to a fibroblastic morphology after four passages. Surface antigens CD29, CD44, CD73, CD90, but not CD34 and CD45, were expressed in all passages. RUNX2 was expressed in MSLCs cultured in all three feeder-free media, while SOX9 and PPARγ were expressed in MSLCs cultured in only DEF-CS500. MSLCs derived from DEF-CS500, which is a single-cell culture medium, grew at a slightly faster rate than those cultured in other media and expressed early-stage genes for tri-lineage differentiation. Taken together, these findings provide valuable information for generating MSLCs using single-cell culture methods.

Building a Total Bioartificial Heart: Harnessing Nature to Overcome the Current Hurdles.

Engineering a bioartificial heart has become a possibility in part because of the regenerative medicine approaches to repairing or replacing damaged organs that have evolved over the past two decades. With the advent of inducible pluripotent stem cell technology, it is now possible to generate personalized cells that make the concept of autologous tissue engineering imaginable. Scaffolds that provide form, function, and biological cues to cells likewise potentially enable the engineering of biocompatible vascularized solid organs. Decellularized organs or tissue matrices retain organ complexity and structure at the macro and micro scales, contain biologically active molecules that support cell phenotype and function, and are vascularized allowing full thickness tissue generation. There is also dynamic reciprocity between the extracellular matrix and cells, which does not occur with synthetic scaffolds and allows both to evolve as functional need changes, making it a unique scaffold. Yet, building a whole heart from decellularized scaffolds and cells requires delivering hundreds of billions of multiple types of cardiac cells appropriately and providing a milieu where they can survive and mature. We propose a novel type of in vivo organ engineering utilizing pre-clinical models where decellularized hearts are heterotopically transplanted with the intent to harness the capability of the body to at least in part repopulate the scaffold. By adding load and electrical input, possibly via temporary mechanical assistance, we posit that vascular and parenchymal cell maturation can occur. In this study, we implanted porcine decellularized hearts acutely and chronically in living recipients in a heterotopic position. We demonstrated that the surgical procedure is critical to prevent coagulation and to increase graft patency. We also demonstrated that short-term implantation promotes endothelial cell adhesion to the vessel lumens and that long-term implantation also promotes tissue formation with evidence of cardiomyocytes and endothelial cells present within the graft. Utilizing endogenous repair capabilities of the recipient in response to a naked ECM, we allowed the transplanted scaffold to direct host cells-both organizationally and functionally. Thus, the scaffold provided necessary cues for cell organization and remodeling within the transplanted organ. Future work would involve culturing partially recellularized engineered organs in bioreactors where mechanical and electrical stimulation can be controlled to promote organ development and then transplanting these after a minimal level of maturation has been achieved.

Isolation and characterization of endothelial colony-forming cells from mononuclear cells of rat bone marrow

Transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) has been used as a therapeutic strategy for vascular repair. However, it remains controversial whether BM-EPCs exhibit clonal endothelial colony-forming cell (ECFC) capacity, a characteristic of true EPCs. The aim of this study was to isolate and explore the cellular properties of BM-ECFCs. We isolated BM-ECFCs from rat bone marrow with high purity via an optimized method. This approach involved the removal of selective colonies based on the conventional differential adhesive culture method used to isolate ECFCs from peripheral and umbilical cord blood. Our results indicate that primary colony BM-ECFCs display a panel of surface antigen markers consistent with endothelial cells. These BM-ECFCs coexpress CD34, CD133, and VEGFR2 at high levels, and these levels decrease with passaging. These cells have high potential for proliferation, migration, and formation of capillary-like structures on Matrigel, and these abilities are retained during ex vivo expansion. Furthermore, BM-ECFCs cultured with 10% or 20% fetal bovine serum demonstrated two different patterns of spontaneous capillary-like structure formation. These results provide a foundation for isolation of ECFCs from human bone marrow for autologous cell transplantation and tissue engineering applications in the future.

Fetal subcutaneous cells have potential for autologous tissue engineering.

Major congenital malformations affect up to 3% of newborns. Infants with prenatally diagnosed soft tissue defects should benefit from having autologous tissue readily available for surgical implantation in the perinatal period. In this study, we investigate fetal subcutaneous cells as cellular source for tissue engineering. Fetal subcutaneous biopsies were collected from elective terminations at gestational Week 20-21. Cells were isolated, expanded, and characterized in vitro. To determine cell coverage, localization, viability, and proliferation in different constructs, the cells were seeded onto a matrix (small intestine submucosa) or in collagen gel with or without poly(ε-caprolactone) mesh and were kept in culture for up to 8weeks before analysis. Angiogenesis was analysed through a tube-forming assay. Fetal subcutaneous cells could be expanded until 43±3 population doublings, expressed mesenchymal markers, and readily differentiate into adipogenic and osteogenic lineages. The cells showed low adherence to small intestine submucosa and did not migrate deep into the matrix. However, in collagen gels, the cells migrated into the gel and proliferated with sustained viability for up to 8weeks. The cells in the matrices expressed Ki67, CD73, and α-smooth muscle actin but not cytokeratin or CD31. Fetal cells derived from subcutaneous tissue demonstrated favourable characteristics for preparation of autologous tissue transplants before birth. Our study supports the theory that cells could be obtained from the fetus during pregnancy for tissue engineering purposes after birth. In a future clinical situation, autologous transplants could be used for reconstructive surgery in severe congenital malformations.

Stem cell-based strategies in vascular surgery

Critical chronic ischemia in patients with underlying arterial occlusive disease requires vascular reconstructive surgery. The limited supply of suitable small-diameter autologous vascular grafts in many patients and obvious disadvantages of synthetic bypass material demand the development of clinically usable tissue-engineered blood vessel substitutes. Despite substantial progress in the field over the last two decades, their implementation into the clinical routine has been challenging. The limited replicative life span of human adult vascular cells and their slow rate of collagenous matrix production in vitro have posed important problems in the development of mechanically robust and biologically functional engineered grafts. With recent advances in stem cell research, new cell sources for vascular tissue engineering have become available. In particular, the discovery of human induced pluripotent stem (iPS) cells derived from adult differentiated cells, as well as of human multipotent adult mesenchymal stem cells without gene modification requirements and related safety concerns, may advance the development of novel autologous cell-based tissue engineering approaches. Here we discuss recent developments in the field of vascular progenitor cells and opportunities and challenges for the clinical translation of stem cell-engineered vascular tissue substitutes.

Repeated penile girth enhancement with biodegradable scaffolds: Microscopic ultrastructural analysis and surgical benefits.

Autologous tissue engineering using biodegradable scaffolds as a carrier is a well-known procedure for penile girth enhancement. We evaluated a group of previously treated patients with the aim to analyze histomorphometric changes after tissue remodeling and to estimate the benefits of repeated procedure. Between February 2012 and December 2016, a group of 21 patients, aged 22–37 (mean 28.0) years, underwent a repeated penile girth enhancement procedure with biodegradable scaffolds. Procedure included insertion of two poly-lactic-co-glycolic acid scaffolds seeded with laboratory-prepared fibroblasts from scrotal tissue specimens. During this procedure, biopsy specimens of tissue formed after the first surgery were taken for microscopic analysis. The mean follow-up was 38 months. Connective tissue with an abundance of connective tissue fibers, small blood vessels, and inflammatory cells were observed in all analyzed surgically removed tissue. Ultrastructural analysis of these tissue samples discovered the presence of large quantities of collagen fibrils running parallel to each other, forming bundles, with a few widely spread fibroblasts. In total, the mean values of flaccid and erect gain in girth after the second surgery were 1.1 ± 0.4 (range: 0.6–1.7) cm and 1.0 ± 0.3 (range: 0.6–1.5) cm, respectively. Microscopic evaluation of newly formed tissue, induced by autologous tissue engineering using biodegradable scaffolds, showed the presence of vascularized loose connective tissue with an abundance of collagen fibers, fibroblasts, and inflammatory cells, indicating active neovascularization and fibrinogenesis. The benefit of the repeated enhancement procedure was statistically significant.

Optimization of Alginate-Based Encapsulation Utilization For Viability and Stability of The Mesenchymal Stem Cell

In the past few decades, attention and research in the field of stem cell are progressing very rapidly. Hospitals in Indonesia have been using stem cells as an alternative to cure some illnesses like diabetes, heart disease, fractures and joints, dental implants, etc. Currently, adult stem cells can be obtained not only from the spinal cord and peripheral vessels, but also from fat tissues of the human body, where it can be isolated as adherent stem cells (mesenchymal stem cells). Consideration of fat tissue as the source of mesenchymal stem cells (MSCs) for autologous tissue engineering is because they are readily available in abundant quantities through minimal invasive procedures, as well as easily cultured and propagated. It is possible to proliferate and differentiate into the desired direction of the network. Stem cell growth requires conditions to grow such as requiring optimum growing conditions such as an environmental temperature of 37°C and a concentration of 5% CO2. Maintenance of MSCs also requires a subculture process, i.e. the process of moving MSCs from a full culture medium to new media; continuous subculture process can cause changes in MSCs. The viability of stem cells may be disrupted by micro-conditions in wounds such as hypoxia, oxidative stress, and inflammation. Therefore, the purpose of this research was to investigate whether alginate-based encapsulation can increase and maintenance stem cell growth at different temperature by using some concentration of alginate and CaCl2 as the formula. Results shown that alginat with low concentration and CaCl2 100mM is suitable for MSCs growth (as in MTT result shown) at 25°C temperature. This can be due to the MSCs encapsulated can adapt and grow within the alginate microcapsule with low concentration. In addition, the media may also easier to get into the microcapsule alginate.

Skin-Derived Stem Cells for Wound Treatment Using Cultured Epidermal Autografts: Clinical Applications and Challenges.

The human skin fulfills important barrier, sensory, and immune functions—all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products.

Preparation and characterization of acellular adipose tissue matrix using a combination of physical and chemical treatments.

Decellularized adipose extracellular matrix (ECM) has been used in the clinic to support the regeneration of adipose tissues. The methods used to produce adipose tissue ECM scaffolds exhibit distinct effects upon the structural and functional components of the resultant scaffold material. The current study presents an acellular ECM scaffold from human adipose tissues derived using successive physical and chemical treatments, including repeated freeze-thaw cycles followed by centrifugation, polar solvent extraction and enzymatic digestion. Cellular components, including nucleic acids were effectively removed without significant disruption of the morphology or structure of the ECM. The compositions of major ECM components were evaluated, including acid/pepsin soluble collagen, sulfated glycosaminoglycan and laminin. The decellularized ECM exhibited satisfactory mechanical properties. Cell seeding experiments involving human adipose-derived stem cells indicated that the decellularized ECM provided an inductive microenvironment for adipogenesis without the need for exogenous differentiation factors. Higher levels of glycerol-3-phosphate dehydrogenase activity were observed among induced cells in the ECM scaffolds when compared with induced cells in collagen type I scaffolds. In conclusion, the results suggested that the decellularized ECM, containing biological and chemical cues of native human ECM, may be an ideal scaffold material for autologous and allograft tissue engineering.