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issn:1937-335
1.  Nanofiber Orientation and Surface Functionalization Modulate Human Mesenchymal Stem Cell Behavior In Vitro 
Tissue Engineering. Part A  2013;20(1-2):398-409.
Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.
doi:10.1089/ten.tea.2012.0426
PMCID: PMC3875145  PMID: 24020454
2.  Sequential Platelet-Derived Growth Factor–Simvastatin Release Promotes Dentoalveolar Regeneration 
Tissue Engineering. Part A  2013;20(1-2):356-364.
Objectives: Timely augmentation of the physiological events of dentoalveolar repair is a prerequisite for the optimization of the outcome of regeneration. This study aimed to develop a treatment strategy to promote dentoalveolar regeneration by the combined delivery of the early mitogenic factor platelet-derived growth factor (PDGF) and the late osteogenic differentiation factor simvastatin.
Materials and Methods: By using the coaxial electrohydrodynamic atomization technique, PDGF and simvastatin were encapsulated in a double-walled poly(d,l-lactide) and poly(d,l-lactide-co-glycolide) (PDLLA–PLGA) microspheres in five different modes: microspheres encapsulating bovine serum albumin (BB), PDGF alone (XP), simvastatin alone (SB), PDGF-in-core and simvastatin-in-shell (PS), and simvastatin-in-core and PDGF-in-shell (SP). The microspheres were characterized using scanning electronic microscopy, and the in vitro release profile was evaluated. Microspheres were delivered to fill large osteotomy sites on rat maxillae for 14 and 28 days, and the outcome of regeneration was evaluated by microcomputed tomography and histological assessments.
Results: Uniform 20-μm controlled release microspheres were successfully fabricated. Parallel PDGF–simvastatin release was noted in the PS group, and the fast release of PDGF followed by the slow release of simvastatin was noted in the SP group. The promotion of osteogenesis was observed in XP, PS, and SP groups at day 14, whereas the SP group demonstrated the greatest bone fill, trabecular numbers, and thickest trabeculae. Bone bridging was evident in the PS and SP group, with significantly increased osteoblasts in the SP group, and osteoclastic cell recruitment was promoted in all bioactive molecule-treated groups. At day 28, osteogenesis was promoted in all bioactive molecule-treated groups. Initial corticalization was noted in the XP, PS, and SP groups. Osteoblasts appeared to be decreased in all groups, and significantly, a greater osteoclastic cell recruitment was noted in the SB and SP groups.
Conclusions: Both PDGF and simvastatin facilitate dentoalveolar regeneration, and sequential PDGF–simvastatin release (SP group) further accelerated the regeneration process through the enhancement of osteoblastogenesis and the promotion of bone maturation.
doi:10.1089/ten.tea.2012.0687
PMCID: PMC3875146  PMID: 23980713
3.  Mucin Covalently Bonded to Microfibers Improves the Patency of Vascular Grafts 
Tissue Engineering. Part A  2013;20(1-2):285-293.
Due to high incidence of vascular bypass procedures, an unmet need for suitable vessel replacements exists, especially for small-diameter (<6 mm) vascular grafts. Here, we developed a novel, bilayered, synthetic vascular graft of 1-mm diameter that consisted of a microfibrous luminal layer and a nanofibrous outer layer, which was tailored to possess the same mechanical property as native arteries. We then chemically modified the scaffold with mucin, a glycoprotein lubricant on the surface of epithelial tissues, by either passive adsorption or covalent bonding using the di-amino-poly(ethylene glycol) linker to microfibers. Under static and physiological flow conditions, conjugated mucin was more stable than adsorbed mucin on the surfaces. Mucin could slightly inhibit blood clotting, and mucin coating suppressed platelet adhesion on microfibrous scaffolds. In the rat common carotid artery anastomosis model, grafts with conjugated mucin, but not adsorbed mucin, exhibited excellent patency and higher cell infiltration into the graft walls. Mucin, which can be easily obtained from autologous sources, offers a novel method for improving the hemocompatibility and surface lubrication of vascular grafts and many other implants.
doi:10.1089/ten.tea.2013.0060
PMCID: PMC3875147  PMID: 23962121
4.  Hypoxia Promotes CEMP1 Expression and Induces Cementoblastic Differentiation of Human Dental Stem Cells in an HIF-1-Dependent Manner 
Tissue Engineering. Part A  2013;20(1-2):410-423.
Cementum covering the tooth root provides attachment for the tooth proper to the surrounding alveolar bone via non-mineralized periodontal ligament (PDL). Cementum protein 1 (CEMP1) has been shown to induce a cementoblastic phenotype in cementoblast precursors cells of PDL. Oxygen availability is a critical signal for correct development of many tissues; however, its role in tooth root and periodontium development remains poorly understood. In this study, we demonstrated that reduced oxygen tension increased CEMP1 expression, mineral deposition, and alkaline phosphatase activity in human dental stem cells such as PDL stem cells and periapical follicular stem cells. Since an oxemic state is transduced by the transcription factor, hypoxia-inducible factor-1 (HIF-1), we performed experiments to determine whether this protein was responsible for the observed changes. We noted that when HIF-1 was activated by gene introduction or chemically, CEMP1 expression and mineralization increased. In contrast, when HIF-1α was silenced, CEMP1 expression and mineralization did not increase in vitro. Furthermore, we showed for the first time that mouse tooth root and periodontium development occurs partly under hypoxic conditions, particularly at the apical part and latently at the PDL space in vivo. Desferrioxamine, an HIF-1 stimulator, enhances CEMP1 expression in the mouse PDL space, suggesting that hypoxia affects cementogenesis of PDL cells lining the surface of the developing tooth root in an HIF-1-dependent manner. These results suggest that HIF-1 activators may have the ability to stimulate regeneration of the tooth root and cementum formation.
doi:10.1089/ten.tea.2013.0132
PMCID: PMC3875148  PMID: 24117017
5.  The Role of Scaffold Architecture and Composition on the Bone Formation by Adipose-Derived Stem Cells 
Tissue Engineering. Part A  2013;20(1-2):434-444.
Scaffold architecture and composition are crucial parameters determining the initial cell spatial distribution and consequently bone tissue formation. Three-dimensional poly-ɛ-caprolactone (PCL) scaffolds with a 0/90° lay-down pattern were plotted and subjected to (1) an oxygen plasma (PCL O) or (2) a postargon plasma modification with gelatin and fibronectin (PCL Fn). These scaffolds with an open pore structure were compared with more compact scaffolds fabricated by conventional processing techniques: oxidized polylactic acid (LA O) and collagen (COL) scaffolds. Human adipose tissue-derived stem cell/scaffold interactions were studied. The study revealed that the biomimetic surface modification of plotted scaffolds did not increase the seeding efficiency. The proliferation and colonization was superior for PCL Fn in comparison with PCL O. The plotted PCL Fn was completely colonized throughout the scaffold, whereas conventional scaffolds only at the edge. Protein-based scaffolds (PCL Fn and COL) enhanced the differentiation, although plotted scaffolds showed a delay in their differentiation compared with compact scaffolds. In conclusion, protein modification of plotted PCL scaffolds enhances uniform tissue formation, but shows a delayed differentiation in comparison with compact scaffolds. The present study demonstrates that biomimetic PCL scaffolds could serve as a guiding template to obtain a uniform bone tissue formation in vivo.
doi:10.1089/ten.tea.2013.0179
PMCID: PMC3875149  PMID: 23998529
6.  Differential Response of Human Adipose Tissue-Derived Mesenchymal Stem Cells, Dermal Fibroblasts, and Keratinocytes to Burn Wound Exudates: Potential Role of Skin-Specific Chemokine CCL27 
Tissue Engineering. Part A  2013;20(1-2):197-209.
Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell–cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006–9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation factors. Our findings have implications for the choice of cell type (ASC or dermal fibroblast) to be used in regenerative medicine strategies and indicate the importance of taking into account interactions with the wound bed when developing advanced therapies for difficult-to-close cutaneous wounds.
doi:10.1089/ten.tea.2013.0123
PMCID: PMC3875150  PMID: 23980822
7.  Multipotent Mesenchymal Stem Cells from Human Subacromial Bursa: Potential for Cell Based Tendon Tissue Engineering 
Tissue Engineering. Part A  2013;20(1-2):239-249.
Rotator cuff injuries are a common clinical problem either as a result of overuse or aging. Biological approaches to tendon repair that involve use of scaffolding materials or cell-based approaches are currently being investigated. The cell-based approaches are focused on applying multipotent mesenchymal stem cells (MSCs) mostly harvested from bone marrow. In the present study, we focused on characterizing cells harvested from tissues associated with rotator cuff tendons based on an assumption that these cells would be more appropriate for tendon repair. We isolated MSCs from bursa tissue associated with rotator cuff tendons and characterized them for multilineage differentiation in vitro and in vivo. Human bursa was obtained from patients undergoing rotator cuff surgery and cells within were isolated using collagenase and dispase digestion. The cells isolated from the tissues were characterized for osteoblastic, adipogenic, chondrogenic, and tenogenic differentiation in vitro and in vivo. The results showed that the cells isolated from bursa tissue exhibited MSCs characteristics as evidenced by the expression of putative cell surface markers attributed to MSCs. The cells exhibited high proliferative capacity and differentiated toward cells of mesenchymal lineages with high efficiency. Bursa-derived cells expressed markers of tenocytes when treated with bone morphogenetic protein-12 (BMP-12) and assumed aligned morphology in culture. Bursa cells pretreated with BMP-12 and seeded in ceramic scaffolds formed extensive bone, as well as tendon-like tissue in vivo. Bone formation was demonstrated by histological analysis and immunofluorescence for DMP-1 in tissue sections made from the scaffolds seeded with the cells. Tendon-like tissue formed in vivo consisted of parallel collagen fibres typical of tendon tissues. Bursa-derived cells also formed a fibrocartilagenous tissue in the ceramic scaffolds. Taken together, the results demonstrate a new source of MSCs with a high potential for application in tendon repair.
doi:10.1089/ten.tea.2013.0197
PMCID: PMC3875151  PMID: 23865619
8.  Adsorbed Fibrinogen Enhances Production of Bone- and Angiogenic-Related Factors by Monocytes/Macrophages 
Tissue Engineering. Part A  2013;20(1-2):250-263.
Macrophages are phagocytic cells with great importance in guiding multiple stages of inflammation and tissue repair. By producing a large number of biologically active molecules, they can affect the behavior of other cells and events, such as the foreign body response and angiogenesis. Since protein adsorption to biomaterials is crucial for the inflammatory process, we addressed the ability of the pro-inflammatory molecule fibrinogen (Fg) to modulate macrophage behavior toward tissue repair/regeneration. For this purpose, we used chitosan (Ch) as a substrate for Fg adsorption. Freshly isolated human monocytes were seeded on Ch substrates alone or previously adsorbed with Fg, and allowed to differentiate into macrophages for 10 days. Cell adhesion and morphology, formation of foreign body giant cells (FBGC), and secretion of a total of 80 cytokines and growth factors were evaluated. Both substrates showed similar numbers of adherent macrophages along differentiation as compared with RGD-coated surfaces, which were used as positive controls. Fg did not potentiate FBGC formation. In addition, actin cytoskeleton staining revealed the presence of punctuate F-actin with more elongated and interconnecting cells on Ch substrates. Antibody array screening and quantification of inflammation- and wound-healing-related factors indicated an overall reduction in Ch-based substrates versus RGD-coated surfaces. At late times, most inflammatory agents were down-regulated in the presence of Fg, in contrast to growth factor production, which was stimulated by Fg. Importantly, on Ch+Fg substrates, fully differentiated macrophages produced significant amounts of macrophage inflammatory protein-1delta (MIP-1δ), platelet-derived growth factor-BB, bone morphogenetic protein (BMP)-5, and BMP-7 compared with Ch alone. In addition, other important factors involved in bone homeostasis and wound healing, such as growth hormone, transforming growth factor-β3, and insulin-like growth factor-binding proteins, as well as several angiogenic mediators, including endocrine gland-derived vascular endothelial factor, fibroblast growth factor-7, and placental growth factor, were significantly promoted by Fg. This work provides a new perspective on the inflammatory response in the context of bone repair/regeneration mediated by a pro-inflammatory protein (Fg) adsorbed onto a biomaterial (Ch) that does not otherwise exhibit osteogenic properties.
doi:10.1089/ten.tea.2012.0439
PMCID: PMC3875152  PMID: 23937279
9.  Beyond Burst Pressure: Initial Evaluation of the Natural History of the Biaxial Mechanical Properties of Tissue-Engineered Vascular Grafts in the Venous Circulation Using a Murine Model 
Tissue Engineering. Part A  2013;20(1-2):346-355.
We previously developed and validated a murine model for investigating neotissue formation in tissue-engineered vascular grafts (TEVGs). Herein, we present the first longitudinal assessment of both the microstructural composition and the mechanical properties of a TEVG through the process of neovessel formation (total scaffold degradation). We show that when (poly)glycolic acid-based biodegradable scaffolds were used as inferior vena cava interposition grafts in mice, the evolving neovessel developed biaxial properties that approached those of the native vein within 24 weeks of implantation. Further, we found that these changes in biaxial properties related temporally to extracellular matrix production and remodeling, including deposition of collagen (types I and III), elastic fibers (elastin and fibrillin-1), and glycosaminoglycans in addition to changes in matrix metalloproteinase (MMP)-2 and -9 activity. Improving our understanding of the mechanobiological principles underlying vascular neotissue formation in TEVGs holds great promise for improving the design of TEVGs and enabling us to continue the translation of this technology from the bench to the clinic.
doi:10.1089/ten.tea.2012.0613
PMCID: PMC3875183  PMID: 23957852
10.  Cyclic Tensile Strain Enhances Osteogenesis and Angiogenesis in Mesenchymal Stem Cells from Osteoporotic Donors 
Tissue Engineering. Part A  2013;20(1-2):67-78.
We have shown that the uniaxial cyclic tensile strain of magnitude 10% promotes and enhances osteogenesis of human mesenchymal stem cells (hMSC) and human adipose-derived stem cells (hASC) from normal, nonosteoporotic donors. In the present study, MSC from osteoporotic donors were analyzed for changes in mRNA expression in response to 10% uniaxial tensile strain to identify potential mechanisms underlying the use of this mechanical loading paradigm for prevention and treatment of osteoporosis. Human MSC isolated from three female, postmenopausal osteoporotic donors were analyzed for their responses to mechanical loading using microarray analysis of over 47,000 gene probes. Human MSC were seeded in three-dimensional collagen type I constructs to mimic the organic extracellular matrix of bone and 10% uniaxial cyclic tensile strain was applied to promote osteogenesis. Seventy-nine genes were shown to be regulated within hMSC from osteoporotic donors in response to 10% cyclic tensile strain. Upregulation of six genes were further confirmed with real-time RT-PCR: jun D proto-oncogene (JUND) and plasminogen activator, urokinase receptor (PLAUR), two genes identified as potential key molecules from network analysis; phosphoinositide-3-kinase, catalytic, delta polypeptide (PIK3CD) and wingless-type MMTV integration site family, member 5B (WNT5B), two genes with known importance in bone biology; and, PDZ and LIM domain 4 (PDLIM4) and vascular endothelial growth factor A (VEGFA), two genes that we have previously shown are significantly regulated in hASC in response to this mechanical stimulus. Function analysis indicated that 10% cyclic tensile strain induced expression of genes associated with cell movement, cell proliferation, and tissue development, including development in musculoskeletal and cardiovascular systems. Our results demonstrate that hMSC from aged, osteoporotic donors are capable of enhanced osteogenic differentiation in response to 10% cyclic tensile strain with significant increases in the expression of genes associated with enhanced cell proliferation, musculoskeletal development, and angiogenesis. Surprisingly, cyclic tensile strain of magnitude 10% not only enhanced osteogenesis in hMSC from osteoporotic donors, but also enhanced expression of angiogenic factors. Better understanding and methodologies to promote osteogenesis in hMSC from elderly, osteoporotic donors may greatly facilitate achieving long-term success in bone regeneration and functional bone tissue engineering for this ever-growing patient population.
doi:10.1089/ten.tea.2013.0006
PMCID: PMC3875187  PMID: 23927731
11.  Influence of Cartilage Extracellular Matrix Molecules on Cell Phenotype and Neocartilage Formation 
Tissue Engineering. Part A  2013;20(1-2):264-274.
Interaction between chondrocytes and the cartilage extracellular matrix (ECM) is essential for maintaining the cartilage's role as a low-friction and load-bearing tissue. In this study, we examined the influence of cartilage zone-specific ECM on human articular chondrocytes (HAC) in two-dimensional and three-dimensional (3D) environments. Two culture systems were used. SYSTEM 1: HAC were cultured on cell-culture plates that had been precoated with the following ECM molecules for 7 days: decorin, biglycan, tenascin C (superficial zone), collagen type II, hyaluronan (HA) (middle and deep zones), and osteopontin (deep zone). Uncoated standard culture plates were used as controls. Expanded cells were examined for phenotypic changes using real-time polymerase chain reaction. In addition, expanded cells were placed into high-density pellet cultures for 14 days. Neocartilage formation was assessed via gene expression and histology evaluations. SYSTEM 2: HAC that were cultured on untreated plates and encapsulated in a 3D alginate scaffold were mixed with one of the zone-specific ECM molecules. Cell viability, gene expression, and histology assessments were conducted on 14-day-old tissues. In HAC monolayer culture, exposure to decorin, HA, and osteopontin increased COL2A1 and aggrecan messenger RNA (mRNA) levels compared with controls. Biglycan up-regulated aggrecan without a significant impact on COL2A1 expression; Tenascin C reduced COL2A1 expression. Neocartilage formed after preculture on tenascin C and collagen type II expressed higher COL2A1 mRNA compared with control pellets. Preculture of HAC on HA decreased both COL2A1 and aggrecan expression levels compared with controls, which was consistent with histology. Reduced proteoglycan 4 (PRG4) mRNA levels were observed in HAC pellets that had been precultured with biglycan and collagen type II. Exposing HAC to HA directly in 3D-alginate culture most effectively induced neocartilage formation, showing increased COL2A1 and aggrecan, and reduced COL1A1 compared with controls. Decorin treatments increased HAC COL2A1 mRNA levels. These data indicate that an appropriate exposure to cartilage-specific ECM proteins could be used to enhance cartilage formation and to even induce the formation of zone-specific phenotypes to improve cartilage regeneration.
doi:10.1089/ten.tea.2012.0618
PMCID: PMC3875188  PMID: 23962090
12.  Effects of Biologic Scaffolds on Human Stem Cells and Implications for CNS Tissue Engineering 
Tissue Engineering. Part A  2013;20(1-2):313-323.
Biologic scaffolds composed of mammalian extracellular matrix (ECM) promote constructive remodeling of tissues via mechanisms that include the recruitment of endogenous stem/progenitor cells, modulation of the host innate immune response, and influence of cell fate differentiation. Such scaffold materials are typically prepared by decellularization of source tissues and are prepared as sheets, powder, or hydrogels. It is plausible that ECM derived from an anatomically distinct tissue would have unique or specific effects on cells that naturally reside in this same tissue. The present study investigated the in vitro effect of a soluble form of ECM derived from central nervous system (CNS) tissue, specifically the spinal cord or brain, versus ECM derived from a non-CNS tissue; specifically, the urinary bladder on the behavior of neural stem cells (NSCs) and perivascular stem cells. All forms of ECM induce positive, mitogenic, and chemotactic effects at concentrations of approximately 100 μg/mL without affecting stem cell viability. CNS-derived ECMs also showed the ability to differentiate NSCs into neurons as indicted by βIII-tubulin expression in two-dimensional culture and neurite extension on the millimeter scale after 24 days of three-dimensional cultures in an ECM hydrogel. These results suggest that solubilized forms of ECM scaffold materials may facilitate the postinjury healing response in CNS tissues.
doi:10.1089/ten.tea.2013.0186
PMCID: PMC3875189  PMID: 24004192
13.  Odontogenic Induction of Dental Stem Cells by Extracellular Matrix-Inspired Three-Dimensional Scaffold 
Tissue Engineering. Part A  2013;20(1-2):92-102.
Currently, root canal therapy is the only clinical treatment available to treat damaged or necrotic dental pulp tissue arising from caries. This treatment results in the loss of tooth vitality. Somatic dental stem cell-based tissue engineering approaches can alleviate this problem by preserving tooth vitality. Dental stem cells are multipotent and under appropriate conditions could be used for dental pulp tissue engineering. Successful use of these cells in pulp repair requires a combination of growth factors and appropriate scaffolds to induce cell differentiation. In this study, we demonstrate the odontogenic differentiation of human dental pulp stem cells (DPSCs) and the human periodontal ligament stem cells when cultured on a decellularized 3D extracellular matrix (ECM) scaffold without the need for exogenous addition of growth factors. Subcutaneous implantation of the ECM scaffolds containing DPSCs showed the formation of dental pulp-like tissue with cells expressing dentin sialoprotein (DSP) and dentin phosphophoryn (DPP). Additionally, we also show that the ECM scaffold can be exploited as a tool to study the extracellular function of multifunctional proteins. These promising results demonstrate the feasibility of developing these biomimetic scaffolds for treatment of dental caries.
doi:10.1089/ten.tea.2013.0192
PMCID: PMC3875192  PMID: 23859633
14.  Quantifying the Biomechanics of Conception: L-Selectin-Mediated Blastocyst Implantation Mechanics with Engineered “Trophospheres” 
Tissue Engineering. Part A  2013;20(1-2):189-196.
An estimated 12% of women in the United States suffer from some form of infertility. In vitro fertilization (IVF) is the most common treatment for infertility encompassing over 99% of all assisted reproductive technologies. However, IVF has a low success rate. Live birth rates using IVF can range from 40% in women younger than 35 years to 4% in women older than 42 years. Costs for a successful IVF outcome can be upward of $61,000. The low success rate of IVF has been attributed to the inability of the blastocyst to implant to the uterus. Blastocyst implantation is initiated by L-selectin expressing cells, trophoblasts, binding to L-selectin ligands, primarily sialyl Lewis X (sLeX), on the uterine surface endometrium. Legal and ethical considerations have limited the research on human subjects and tissues, whereas animal models are costly or do not properly mimic human implantation biochemistry. In this work, we describe a cellular model system for quantifying L-selectin adhesion mechanics. L-selectin expression was confirmed in Jeg-3, JAR, and BeWo cell lines, with only Jeg-3 cells exhibiting surface expression. Jeg-3 cells were cultured into three-dimensional spheres, termed “trophospheres,” as a mimic to human blastocysts. Detachment assays using a custom-built parallel plate flow chamber show that trophospheres detach from sLeX functionalized slides with 2.75×10−3 dyn of force and 7.5×10−5 dyn-cm of torque. This work marks the first time a three-dimensional cell model has been utilized for quantifying L-selectin binding mechanics related to blastocyst implantation.
doi:10.1089/ten.tea.2013.0067
PMCID: PMC3875198  PMID: 23927766
15.  Balancing the Rates of New Bone Formation and Polymer Degradation Enhances Healing of Weight-Bearing Allograft/Polyurethane Composites in Rabbit Femoral Defects 
Tissue Engineering. Part A  2013;20(1-2):115-129.
There is a compelling clinical need for bone grafts with initial bone-like mechanical properties that actively remodel for repair of weight-bearing bone defects, such as fractures of the tibial plateau and vertebrae. However, there is a paucity of studies investigating remodeling of weight-bearing bone grafts in preclinical models, and consequently there is limited understanding of the mechanisms by which these grafts remodel in vivo. In this study, we investigated the effects of the rates of new bone formation, matrix resorption, and polymer degradation on healing of settable weight-bearing polyurethane/allograft composites in a rabbit femoral condyle defect model. The grafts induced progressive healing in vivo, as evidenced by an increase in new bone formation, as well as a decrease in residual allograft and polymer from 6 to 12 weeks. However, the mismatch between the rates of autocatalytic polymer degradation and zero-order (independent of time) new bone formation resulted in incomplete healing in the interior of the composite. Augmentation of the grafts with recombinant human bone morphogenetic protein-2 not only increased the rate of new bone formation, but also altered the degradation mechanism of the polymer to approximate a zero-order process. The consequent matching of the rates of new bone formation and polymer degradation resulted in more extensive healing at later time points in all regions of the graft. These observations underscore the importance of balancing the rates of new bone formation and degradation to promote healing of settable weight-bearing bone grafts that maintain bone-like strength, while actively remodeling.
doi:10.1089/ten.tea.2012.0762
PMCID: PMC3875200  PMID: 23941405
16.  Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering 
Tissue Engineering. Part A  2013;20(1-2):210-224.
Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25×106 cells/mL, containing an estimated 5×104 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2×105 cells/mL) were added to a 65–35 wt% collagen-chitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O2 (normoxia) or 5% O2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMC-microbeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using culture-expanded MSC for bone tissue engineering.
doi:10.1089/ten.tea.2013.0151
PMCID: PMC3875201  PMID: 23879621
17.  In Vivo Bone Regeneration Using Tubular Perfusion System Bioreactor Cultured Nanofibrous Scaffolds 
Tissue Engineering. Part A  2013;20(1-2):139-146.
The use of bioreactors for the in vitro culture of constructs for bone tissue engineering has become prevalent as these systems may improve the growth and differentiation of a cultured cell population. Here we utilize a tubular perfusion system (TPS) bioreactor for the in vitro culture of human mesenchymal stem cells (hMSCs) and implant the cultured constructs into rat femoral condyle defects. Using nanofibrous electrospun poly(lactic-co-glycolic acid)/poly(ɛ-caprolactone) scaffolds, hMSCs were cultured for 10 days in vitro in the TPS bioreactor with cellular and acellular scaffolds cultured statically for 10 days as a control. After 3 and 6 weeks of in vivo culture, explants were removed and subjected to histomorphometric analysis. Results indicated more rapid bone regeneration in defects implanted with bioreactor cultured scaffolds with a new bone area of 1.23±0.35 mm2 at 21 days compared to 0.99±0.43 mm2 and 0.50±0.29 mm2 in defects implanted with statically cultured scaffolds and acellular scaffolds, respectively. At the 21 day timepoint, statistical differences (p<0.05) were only observed between defects implanted with cell containing scaffolds and the acellular control. After 42 days, however, defects implanted with TPS cultured scaffolds had the greatest new bone area with 1.72±0.40 mm2. Defects implanted with statically cultured and acellular scaffolds had a new bone area of 1.26±0.43 mm2 and 1.19±0.33 mm2, respectively. The increase in bone growth observed in defects implanted with TPS cultured scaffolds was statistically significant (p<0.05) when compared to both the static and acellular groups at this timepoint. This study demonstrates the efficacy of the TPS bioreactor to improve bone tissue regeneration and highlights the benefits of utilizing perfusion bioreactor systems to culture MSCs for bone tissue engineering.
doi:10.1089/ten.tea.2013.0168
PMCID: PMC3875202  PMID: 23865551
18.  Chondrogenesis of Mesenchymal Stem Cells in an Osteochondral Environment Is Mediated by the Subchondral Bone 
Tissue Engineering. Part A  2013;20(1-2):23-33.
In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGFβ) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFβ; however, the observed results could not be attributed to TGFβ, as either capturing TGFβ with an antibody or blocking the canonical TGFβ signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFβ family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.
doi:10.1089/ten.tea.2013.0080
PMCID: PMC3875203  PMID: 23980750
19.  Building an Anisotropic Meniscus with Zonal Variations 
Tissue Engineering. Part A  2013;20(1-2):294-302.
Toward addressing the difficult problems of knee meniscus regeneration, a self-assembling process has been used to re-create the native morphology and matrix properties. A significant problem in such attempts is the recapitulation of the distinct zones of the meniscus, the inner, more cartilaginous and the outer, more fibrocartilaginous zones. In this study, an anisotropic and zonally variant meniscus was produced by self-assembly of the inner meniscus (100% chondrocytes) followed by cell seeding the outer meniscus (coculture of chondrocytes and meniscus cells). After 4 weeks in culture, the engineered, inner meniscus exhibited a 42% increase in both instantaneous and relaxation moduli and a 62% increase in GAG/DW, as compared to the outer meniscus. In contrast, the circumferential tensile modulus and collagen/DW of the outer zone was 101% and 129% higher, respectively, than the values measured for the inner zone. Furthermore, there was no difference in the radial tensile modulus between the control and zonal engineered menisci, suggesting that the inner and outer zones of the engineered zonal menisci successfully integrated. These data demonstrate that not only can biomechanical and biochemical properties be engineered to differ by the zone, but they can also recapitulate the anisotropic behavior of the knee meniscus.
doi:10.1089/ten.tea.2013.0098
PMCID: PMC3875205  PMID: 23931258
20.  Characterization of Total and Active Matrix Metalloproteinases-1, -3, and -13 Synthesized and Secreted by Anterior Cruciate Ligament Fibroblasts in Three-Dimensional Collagen Gels 
Tissue Engineering. Part A  2013;20(1-2):171-177.
Anterior cruciate ligament (ACL) injury and subsequent reconstructive surgery is increasing with an estimated 200,000 reconstructions performed yearly in the United States. Current treatment requires reconstruction with autograft or allograft tissue with inherent disadvantages. The development of tissue-engineered ligament replacements or scaffolds may provide an alternative treatment method minimizing these issues. The study of ligament fibroblast catabolic and anabolic responses to mechanical and biologic stimuli in three-dimensional (3D) cell culture systems is critical to the development of such therapies. A 3D cell culture system was used to measure the total content and active forms of matrix metalloproteinases (MMPs)-1, -3, and -13 to assess the potential role of the mechanical environment in regulation of matrix turnover by ligament fibroblasts. The production, retention, and secretion of MMPs by ACL fibroblasts in 3D culture were measured over a 14-day period. The total MMP content and MMP activity were determined. The level of all MMPs studied increased over 7–10 days and then reached a steady state or decreased slightly in both the collagen gels and the media. This system will now permit the study of externally applied cyclic and static strains, strain deprivation, and the potential combined role of the cytoskeleton and MMPs in matrix turnover in ligaments.
doi:10.1089/ten.tea.2012.0669
PMCID: PMC3875206  PMID: 23879595
21.  The Effect of Nanotopography on Modulating Protein Adsorption and the Fibrotic Response 
Tissue Engineering. Part A  2013;20(1-2):130-138.
Understanding and modulating the cellular response to implanted biomaterials is crucial for the field of tissue engineering and regenerative medicine. Since cells typically reside in an extracellular matrix containing nanoscale architecture, identifying synthetic nanostructures that induce desirable cellular behaviors could greatly impact the field. Using nanoimprint lithography, nanostructured patterns were generated on thin film polymeric materials. The ability of these surfaces to influence protein adsorption, fibroblast proliferation and morphology, and fibrotic markers was investigated. Nanostructured features with aspect ratios greater than five allowed for less protein adsorption, resulting in decreased fibroblast proliferation and rounded cellular morphology. These nanofeatures also induced significantly lower gene expression of collagen 1α2, collagen 3α1, and growth factors such as connective tissue growth factor, integrin linked kinase, transforming growth factor β1 (TGF-β1), and epidermal growth factor, key factors associated with a fibrotic response. The results demonstrate that select nanostructured surfaces could be used to modulate the fibrotic behavior in cells and have the potential to be used as antifibrotic architecture for medical implants or tissue engineering scaffolds.
doi:10.1089/ten.tea.2012.0772
PMCID: PMC3875207  PMID: 23914986
22.  Extracellular Matrices Decellularized from Embryonic Stem Cells Maintained Their Structure and Signaling Specificity 
Tissue Engineering. Part A  2013;20(1-2):54-66.
Embryonic stem cells (ESCs) emerge as a promising tool for tissue engineering and regenerative medicines due to their extensive self-renewal ability and the capacity to give rise to cells from all three-germ layers. ESCs also secrete a large amount of endogenous extracellular matrices (ECMs), which play an important role in regulating ESC self-renewal, lineage commitment, and tissue morphogenesis. ECMs derived from ESCs have a broader signaling capacity compared to somatic ECMs and are predicted to have a lower risk of tumor formation associated with ESCs. In this study, ECMs from undifferentiated ESC monolayers, undifferentiated aggregates, or differentiated embryoid bodies at different developmental stages and lineage specifications were decellularized and their capacities to direct ESC proliferation and differentiation were characterized. The results demonstrate that the ESC-derived ECMs were able to influence ESC proliferation and differentiation by direct interactions with the cells and by influencing the signaling functions of the regulatory macromolecules such as retinoic acid. Such matrices have the potential to present regulatory signals to direct lineage- and development-specific cellular responses for in vitro applications or cell delivery.
doi:10.1089/ten.tea.2012.0690
PMCID: PMC3875208  PMID: 23848515
23.  Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model 
Tissue Engineering. Part A  2013;20(1-2):225-238.
In vivo, epithelial cells are connected both anatomically and functionally with stromal keratocytes. Co-culturing aims at recapturing this cellular anatomy and functionality by bringing together two or more cell types within the same culture environment. Corneal stromal cells were activated to their injury phenotype (fibroblasts) and expanded before being encapsulated in type I collagen hydrogels constructs. Three different epithelial-stromal co-culture methods were then examined: epithelial explant; transwell; and the use of conditioned media. The aim was to determine whether the native, inactivated keratocyte cell phenotype could be restored in vitro. Media supplementation with transforming growth factor beta-1 (TGF-β1) was then used to determine whether the inactivated stromal cells retained their plasticity in vitro and could be re-activated to the fibroblast phenotype. Finally, media supplementation with wortmannin was used to inhibit epithelial–stromal cell interactions. Two different nondestructive techniques, spherical indentation and optical coherence tomography, were used to reveal how epithelial-stromal co-culturing with TGF-β1, and wortmannin media supplementation, respectively, affect stromal cell behavior and differentiation in terms of construct contraction and elastic modulus measurement. Cell viability, phenotype, morphology, and protein expression were investigated to corroborate our mechanical findings. It was shown that activated stromal cells could be inactivated to a keratocyte phenotype via co-culturing and that they retained their plasticity in vitro. Activated corneal stromal cells that were fibroblastic in phenotype were successfully reverted to a nonactivated keratocyte cell lineage in terms of behavior and biological properties; and then back again via TGF-β1 media supplementation. It was then revealed that epithelial–stromal interactions can be blocked via the use of wortmannin inhibition. A greater understanding of stromal–epithelial interactions and what mediates them offers great pharmacological potential in the regulation of corneal wound healing, with the potential to treat corneal diseases and injury by which such interactions are vital.
doi:10.1089/ten.tea.2013.0167
PMCID: PMC3875209  PMID: 23895175
24.  Development and In Vivo Evaluation of Small-Diameter Vascular Grafts Engineered by Outgrowth Endothelial Cells and Electrospun Chitosan/Poly(ɛ-Caprolactone) Nanofibrous Scaffolds 
Tissue Engineering. Part A  2013;20(1-2):79-91.
Successful engineering of a small-diameter vascular graft is still a challenge despite numerous attempts for decades. The present study aimed at developing a tissue-engineered vascular graft (TEVG) using autologous outgrowth endothelial cells (OECs) and a hybrid biodegradable polymer scaffold. OECs were harvested from canine peripheral blood and proliferated in vitro, as well as identified by immunofluorescent staining. Electrospun hybrid chitosan/poly(ɛ-caprolactone) (CS/PCL) nanofibers were fabricated and served as vascular scaffolds. TEVGs were constructed in vitro by seeding OECs onto CS/PCL scaffolds, and then implanted into carotid arteries of cell-donor dogs (n=6). After 3 months of implantation, 5 out of 6 of TEVGs remained patent as compared with 1 out of 6 of unseeded grafts kept patent. Histological and immunohistochemical analyses of the TEVGs retrieved at 3 months revealed the regeneration of endothelium, and the presence of collagen and elastin. OECs labeled with fluorescent dye before implantation were detected in the retrieved TEVGs, indicating that the OECs participated in the vascular tissue regeneration. Biomechanical testing of TEVGs showed good mechanical properties that were closer to native carotid arteries. RT-PCR and western blot analysis demonstrated that TEVGs had favorable biological functional properties resembling native arteries. Overall, this study provided a new strategy to develop small-diameter TEVGs with excellent biocompatibility and regeneration ability.
doi:10.1089/ten.tea.2013.0020
PMCID: PMC3875210  PMID: 23902162
25.  Fluorapatite Enhances Mineralization of Mesenchymal/Endothelial Cocultures 
Tissue Engineering. Part A  2013;20(1-2):12-22.
In addition to the widely used mesenchymal stem cells (MSCs), endothelial cells appear to be a favorable cell source for hard tissue regeneration. Previously, fluorapatite was shown to stimulate and enhance mineralization of MSCs. This study aims to investigate the growth of endothelial cells on synthesized ordered fluorapatite surfaces and their effect on the mineralization of adipose-derived stem cells (ASCs) through coculture. Endothelial cells were grown on fluorapatite surfaces and characterized by cell counting, flow cytometry, scanning electron microscopy, and enzyme-linked immunosorbent assay (ELISA). Cells were then cocultured with ASCs and stained for alkaline phosphatase and mineral formation. Fibroblast growth factor (FGF) pathway perturbation and basic FGF (bFGF) treatment of the ASCs were also conducted to observe their effects on differentiation and mineralization of these cells. Fluorapatite surfaces showed good biocompatibility in supporting endothelial cells. Without a mineralization supplement, coculture with endothelial cells induced osteogenic differentiation of ASCs, which was further enhanced by the fluorapatite surfaces. This suggested a combined stimulating effect of endothelial cells and fluorapatite surfaces on the enhanced mineralization of ASCs. Greater amounts of bFGF release by endothelial cells alone or cocultures with ASCs stimulated by fluorapatite surfaces, together with FGF pathway perturbation and bFGF treatment results, suggested that the FGF signaling pathway may function in this process.
doi:10.1089/ten.tea.2013.0113
PMCID: PMC3875212  PMID: 23859365

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