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1.  Osteogenic Differentiation of Adipose-Derived Stem Cells Is Hypoxia-Inducible Factor-1 Independent 
Tissue Engineering. Part A  2013;19(13-14):1583-1591.
Tissue engineering is a promising approach to repair critical-size defects in bone. Damage to vasculature at the defect site can create a lower O2 environment compared with healthy bone. Local O2 levels influence stem cell behavior, as O2 is not only a nutrient, but also a signaling molecule. The hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates a wide range of O2-related genes and its contribution in bone repair/formation is an important area that can be exploited. In this study, we examined the effect of low O2 environments (1% and 2% O2) on the osteogenic differentiation of adipose-derived stem cells in both two-dimensional (2-D) and three-dimensional (3-D) culture systems. To determine the role of HIF-1 in the differentiation process, an inhibitor was used to block the HIF-1 activity. The samples were examined for osteogenesis markers as measured by quantification of the alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteonectin (ON) and osteopontin (OPN). Results show a downregulation of the osteogenic markers (ALP activity, mineralization, ON, OPN) in both 1% and 2% O2 when compared to 20% O2 in both 2-D and 3-D culture. Vascular endothelial growth factor secretion over 28 days was significantly higher in low O2 environments and HIF-1 inhibition reduced this effect. The inhibition of the HIF-1 activity did not have a significant impact on the expression of the osteogenic markers, suggesting HIF-1-independent inhibition of osteogenic differentiation in hypoxic conditions.
PMCID: PMC3665297  PMID: 23394201
2.  Collagen: Finding a Solution for the Source 
Tissue Engineering. Part A  2013;19(13-14):1491-1494.
Extracellular matrix (ECM)-based scaffolds, through their inherent bioactivity and molecular recognition signals, provide the ideal substrate for tissue engineering and regenerative applications. Collagen, the most abundant ECM protein, has proven itself to be a very versatile material with applications in many fields, including the leather and food industries, cosmetics, drug delivery, and tissue engineering. However, doubts persist about the optimal source of collagen for tissue engineering applications, given possible immunogenicity and disease transmission associated with animal sources and reduced bioactivity and availability of recombinant technologies. In this special edition, an attempt is made to elucidate the advantages of plant-derived human recombinant collagen and its applications in tissue engineering, particularly skin and wound healing. While results are promising, the widespread use of animal-derived collagen means that recombinant technologies may find applications in niche areas.
PMCID: PMC3665299  PMID: 23240852
3.  Shining Light on Collagen: Expressing Collagen in Plants 
Tissue Engineering. Part A  2013;19(13-14):1499-1501.
Collagens are a remarkable group of proteins that are critical from a physiological perspective due to their diverse and versatile functions in vivo. However, collagens are challenging to generate ex vivo for biomaterials or regenerative medicine due to their complex processing and assembly into functional materials. Therefore, collagen availability remains a major unmet need for biomaterials, as relatively limited supplies of the protein in pure form are available mainly through harvesting bovine tissues. This animal source, subsequent to purification, remains associated with significant safety concerns due to the potential carryover of animal-derived diseases. Other more limited sources of animal collagens are also commercially available, as well as collagens generated in heterologous hosts; however, the challenge to these sources remains both economic and structural. The need for new safe sources of collagens remains high, with a significant potential impact in areas of medicine when considering the opportunity to mimic native collagen features. The articles in this issue of the journal focus on plant-derived collagens to address some of these needs. Progress toward plant production of collagens, the ability to self-assemble these recombinant proteins into higher-order structures, and the utility of these materials in various medical applications suggest an important path forward for the field.
PMCID: PMC3665302  PMID: 23521064
4.  Absence of Immune Responses with Xenogeneic Collagen and Elastin 
Tissue Engineering. Part A  2013;19(13-14):1592-1600.
Novel tissue-engineering approaches for cardiovascular matrices based on xenogeneic extracellular matrix protein (ECMp) constituents require a detailed evaluation of their interaction with essential immune cell subsets playing a role in innate or adaptive immunity. Therefore, in this study, the effects of xenogeneic (porcine, bovine) collagen type I and elastin as the two main components of the heart valve ECM were analyzed in comparison to their human equivalents. First, their potential to induce maturation and cytokine secretion of human dendritic cells (DC) was tested by flow cytometry. Second, the influence on proliferation and cytokine release of purified human B and T cells was measured. We could demonstrate that xenogeneic collagen type I and elastin are not able to trigger the maturation of DC as verified by the lack of CD83 induction accompanied by a low tumor necrosis factor-α release. Moreover, both ECMp showed no effect on the proliferation and the interleukin-6 release of either unstimulated or prestimulated B cells. Additionally, anti-CD3-induced purified T cell proliferation and secretion of cytokines was not affected. All in vitro data verify the low immunogenicity of porcine and bovine collagen type I and elastin and favor their suitability for tissue-engineered scaffolds.
PMCID: PMC3665304  PMID: 23406399
5.  Cutaneous Wound Healing After Treatment with Plant-Derived Human Recombinant Collagen Flowable Gel 
Tissue Engineering. Part A  2013;19(13-14):1519-1526.
Chronic wounds, particularly diabetic ulcers, represent a main public health concern with significant costs. Ulcers often harbor an additional obstacle in the form of tunneled or undermined wounds, requiring treatments that can reach the entire wound tunnel, because bioengineered grafts are typically available only in a sheet form. While collagen is considered a suitable biodegradable scaffold material, it is usually extracted from animal and human cadaveric sources, and accompanied by potential allergic and infectious risks. The purpose of this study was to test the performance of a flowable gel made of human recombinant type I collagen (rhCollagen) produced in transgenic tobacco plants, indicated for the treatment of acute, chronic, and tunneled wounds. The performance of the rhCollagen flowable gel was tested in an acute full-thickness cutaneous wound-healing rat model and compared to saline treatment and two commercial flowable gel control products made of bovine collagen and cadaver human skin collagen. When compared to the three control groups, the rhCollagen-based gel accelerated wound closure and triggered a significant jumpstart to the healing process, accompanied by enhanced re-epithelialization. In a cutaneous full-thickness wound pig model, the rhCollagen-based flowable gel induced accelerated wound healing compared to a commercial product made of bovine tendon collagen. By day 21 post-treatment, 95% wound closure was observed with the rhCollagen product compared to 68% closure in wounds treated with the reference product. Moreover, rhCollagen treatment induced an early angiogenic response and induced a significantly lower inflammatory response than in the control group. In summary, rhCollagen flowable gel proved to be efficacious in animal wound models and is expected to be capable of reducing the healing time of human wounds.
PMCID: PMC3665308  PMID: 23259631
6.  Plant-Derived Human Collagen Scaffolds for Skin Tissue Engineering 
Tissue Engineering. Part A  2013;19(13-14):1507-1518.
Tissue engineering scaffolds are commonly formed using proteins extracted from animal tissues, such as bovine hide. Risks associated with the use of these materials include hypersensitivity and pathogenic contamination. Human-derived proteins lower the risk of hypersensitivity, but possess the risk of disease transmission. Methods engineering recombinant human proteins using plant material provide an alternate source of these materials without the risk of disease transmission or concerns regarding variability. To investigate the utility of plant-derived human collagen (PDHC) in the development of engineered skin (ES), PDHC and bovine hide collagen were formed into tissue engineering scaffolds using electrospinning or freeze-drying. Both raw materials were easily formed into two common scaffold types, electrospun nonwoven scaffolds and lyophilized sponges, with similar architectures. The processing time, however, was significantly lower with PDHC. PDHC scaffolds supported primary human cell attachment and proliferation at an equivalent or higher level than the bovine material. Interleukin-1 beta production was significantly lower when activated THP-1 macrophages where exposed to PDHC electrospun scaffolds compared to bovine collagen. Both materials promoted proper maturation and differentiation of ES. These data suggest that PDHC may provide a novel source of raw material for tissue engineering with low risk of allergic response or disease transmission.
PMCID: PMC3665310  PMID: 23298216
7.  Glycosaminoglycan Mimetic Associated to Human Mesenchymal Stem Cell-Based Scaffolds Inhibit Ectopic Bone Formation, but Induce Angiogenesis In Vivo 
Tissue Engineering. Part A  2013;19(13-14):1641-1653.
Tissue engineering approaches to stimulate bone formation currently combine bioactive scaffolds with osteocompetent human mesenchymal stem cells (hMSC). Moreover, osteogenic and angiogenic factors are required to promote differentiation and survival of hMSC through improved vascularization through the damaged extracellular matrix (ECM). Glycosaminoglycans (GAGs) are ECM compounds acting as modulators of heparin-binding protein activities during bone development and regenerative processes. GAG mimetics have been proposed as ECM stabilizers and were previously described for their positive effects on bone formation and angiogenesis after local treatment. Here, we developed a strategy associating the GAG mimetic [OTR4120] with bone substitutes to optimize stem cell-based therapeutic products. We showed that [OTR4120] was able to potentiate proliferation, migration, and osteogenic differentiation of hMSC in vitro. Its link to tricalcium phosphate/hydroxyapatite scaffolds improved their colonization by hMSC. Surprisingly, when these combinations were tested in an ectopic model of bone formation in immunodeficient mice, the GAG mimetics inhibit bone formation induced by hMSC and promoted an osteoclastic activity. Moreover, the inflammatory response was modulated, and the peri-implant vascularization stimulated. All together, these findings further support the ability of GAG mimetics to organize the local ECM to coordinate the host response toward the implanted biomaterial, and to inhibit the abnormal bone formation process on a subcutaneous ectopic site.
PMCID: PMC3665317  PMID: 23521005
8.  Dynamic Shear Stress Regulation of Inflammatory and Thrombotic Pathways in Baboon Endothelial Outgrowth Cells 
Tissue Engineering. Part A  2013;19(13-14):1573-1582.
Endothelial outgrowth cells (EOCs) have garnered much attention as a potential autologous endothelial source for vascular implants or in tissue engineering applications due to their ease of isolation and proliferative ability; however, how these cells respond to different hemodynamic cues is ill-defined. This study investigates the inflammatory and thrombotic response of baboon EOCs (BaEOCs) to four hemodynamic conditions using the cone and plate shear apparatus: steady, laminar shear stress (SS); pulsatile, nonreversing laminar shear stress (PS); oscillatory, laminar shear stress (OS); and net positive, pulsatile, reversing laminar shear stress (RS). In summary, endothelial nitric oxide synthase (eNOS) mRNA was significantly upregulated by SS compared to OS. No differences were found in the mRNA levels of the inflammatory markers intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) between the shear conditions; however, OS significantly increased the number of monocytes bound when compared to SS. Next, SS increased the anti-thrombogenic mRNA levels of CD39, thrombomodulin, and endothelial protein-C receptor (EPCR) compared to OS. SS also significantly increased CD39 and EPCR mRNA levels compared to RS. Finally, no significant differences were detected when comparing pro-thrombotic tissue factor mRNA or its activity levels. These results indicate that shear stress can have beneficial (SS) or adverse (OS, RS) effects on the inflammatory or thrombotic potential of EOCs. Further, these results suggest SS hemodynamic preconditioning may be optimal in increasing the efficacy of a vascular implant or in tissue-engineered applications that have incorporated EOCs.
PMCID: PMC3665318  PMID: 23406430
9.  Nanoporosity Significantly Enhances the Biological Performance of Engineered Glass Tissue Scaffolds 
Tissue Engineering. Part A  2013;19(13-14):1632-1640.
Nanoporosity is known to impact the performance of implants and scaffolds such as bioactive glass (BG) scaffolds, either by providing a higher concentration of bioactive chemical species from enhanced surface area, or due to inherent nanoscale topology, or both. To delineate the role of these two characteristics, BG scaffolds have been fabricated with nearly identical surface area (81 and 83±2 m2/g) but significantly different pore size (av. 3.7 and 17.7 nm) by varying both the sintering temperature and the ammonia concentration during the solvent exchange phase of the sol-gel fabrication process. In vitro tests performed with MC3T3-E1 preosteoblast cells on such scaffolds show that initial cell attachment is increased on samples with the smaller nanopore size, providing the first direct evidence of the influence of nanopore topography on cell response to a bioactive structure. Furthermore, in vivo animal tests in New Zealand rabbits (subcutaneous implantation) indicate that nanopores promote colonization and cell penetration into these scaffolds, further demonstrating the favorable effects of nanopores in tissue-engineering-relevant BG scaffolds.
PMCID: PMC3665319  PMID: 23427819
10.  Characterization of Schwann Cells in Self-Assembled Sheets from Thermoresponsive Substrates 
Tissue Engineering. Part A  2013;19(13-14):1601-1609.
Schwann cells are the vital glial cells in the development and regeneration of the peripheral nervous system (PNS). Recently, Schwann cell transplantation has emerged as one of the attractive candidates in treating demyelinating diseases resulting from the PNS and central nervous system injuries. Schwann cells are usually injected as cell suspensions or transplanted after being seeded on extracellular matrix proteins or biodegradable polymeric scaffolds. In these approaches, the adherens junctions between Schwann cells present in vivo are not readily replicated as Schwann cells dispersed as individual cells. Here we describe a procedure to grow a large amount of Schwann cells in a sheet architecture that can be either transplanted or injected and provide some insights into the influence of a sheet-like cell organization on the function of Schwann cells, including their viability, proliferation, alignment, and migration. The Schwann cell sheet was successfully generated through coating the culture plate surfaces by layer-by-layer self-assembly of the thermoresponsive polymer poly-(N-isopropylacrylamide) (PNIPAAM). Further characterization of the Schwann cell sheet showed that Schwann cells in sheet were highly viable, but maintained a lower proliferation rate than individual Schwann cells. The levels of nerve growth factor and glial cell-derived neurotrophic factor were also maintained in Schwann cell sheets. The protein level of a cyclin-dependent kinase inhibitor, p27, was upregulated in the Schwann cell sheet. Both alignment with axon-like nanofibers and migration of Schwann cells are not significantly different between Schwann cells in a sheet-like organization and as individual cells. We conclude that Schwann cell sheet engineering presents a promising method for cell-based nerve injury therapy, as well as a model to study the control of Schwann cell proliferation in response to intercellular organization.
PMCID: PMC3665320  PMID: 23477904
11.  Three-Dimensional Cell Culture Environment Promotes Partial Recovery of the Native Corneal Keratocyte Phenotype from a Subcultured Population 
Tissue Engineering. Part A  2013;19(13-14):1564-1572.
Corneal disease is the fourth leading cause of blindness. According to the World Health Organization, roughly 1.6 million people globally are blind as a result of this disease. The only current treatment for corneal opacity is a corneal tissue transplant. Unfortunately, the demand for tissue exceeds supply, making a tissue-engineered in vitro cornea highly desirable. For an in vitro cornea to be useful, it must be transparent, which requires downregulation of the light-scattering intracellular protein alpha-smooth muscle actin (αSMA) and upregulation of the native corneal marker, aldehyde dehydrogenase 1A1 (ALDH1A1). This study focuses on the effects of a three-dimensional (3D) matrix on the expression levels of αSMA and ALDH1A1 by a subcultured population of rabbit corneal keratocytes and the comparison of the 3D matrix effects to other culture conditions. We show that, through western blot and quantitative real-time PCR, the presence of collagen strongly downregulates αSMA. Further, 3D cultures maintain low actin expression even in the presence of a proinflammatory cytokine, transforming growth factor-beta (TGF-β). Finally, 3D culture conditions show a partial recovery of ALDH1A1 expression, which has never been previously observed in a serum-exposed subcultured cell population. Overall, this study suggests that 3D culture is not only a relatively stronger signal than both collagen and TGF-β, it is also sufficient to induce some recovery of ALDH1A1 and the native corneal phenotype despite the presence of serum.
PMCID: PMC3665321  PMID: 23410050
12.  Reconstruction of Abdominal Wall Musculofascial Defects with Small Intestinal Submucosa Scaffolds Seeded with Tenocytes in Rats 
Tissue Engineering. Part A  2013;19(13-14):1543-1553.
The repair of abdominal wall defects following surgery remains a difficult challenge. Although multiple methods have been described to restore the integrity of the abdominal wall, there is no clear consensus on the ideal material for reconstruction. This study explored the feasibility of in vivo reconstruction of a rat model of an abdominal wall defect with a composite scaffold of tenocytes and porcine small intestinal submucosa (SIS). In the current study, we created a 2×1.5 cm abdominal wall defect in the anterolateral abdominal wall of Sprague-Dawley rats, which were assigned into three groups: the cell-SIS construct group, the cell-free SIS scaffold group, and the abdominal wall defect group. Tenocytes were obtained from the tendons of rat limbs. After isolation and expansion, cells (2×107/mL) were seeded onto the three-layer SIS scaffolds and cultured in vitro for 5 days. Cell-SIS constructs or cell-free constructs were implanted to repair the abdominal wall defects. The results showed that the tenocytes could grow on the SIS scaffold and secreted corresponding matrices. In addition, both scaffolds could repair the abdominal wall defects with no hernia recurrence. In comparison to the cell-free SIS scaffold, the composite scaffold exhibited increased vascular regeneration and mechanical strength. Furthermore, following increased time in vivo, the mechanical strength of the composite scaffold became stronger. The results indicate that the composite scaffold can provide increased mechanical strength that may be suitable for repairing abdominal wall defects.
PMCID: PMC3665322  PMID: 23402600
13.  Implantable Three-Dimensional Salivary Spheroid Assemblies Demonstrate Fluid and Protein Secretory Responses to Neurotransmitters 
Tissue Engineering. Part A  2013;19(13-14):1610-1620.
Radiation treatment in patients with head and neck tumors commonly results in hyposalivation and xerostomia due to the loss of fluid-secreting salivary acinar cells. Patients develop susceptibility to oral infections, dental caries, impaired speech and swallowing, reducing the quality of life. Clinical management is largely unsatisfactory. The development of a tissue-engineered, implantable salivary gland will greatly benefit patients suffering from xerostomia. This report compares the ability of a 2.5-dimensional (2.5D) and a three-dimensional (3D) hyaluronic acid (HA)-based culture system to support functional salivary units capable of producing fluid and phenotypic proteins. Parotid cells seeded on 2.5D, as well as those encapsulated in 3D HA hydrogels, self-assembled into acini-like structures and expressed functional neurotransmitter receptors. Structures in 3D hydrogels merged to form organized 50 μm spheroids that could be maintained in culture for over 100 days and merged to form structures over 500 μm in size. Treatment of acini-like structures with the β-adrenergic agonists norepinephrine or isoproterenol increased granule production and α-amylase staining in treated structures, demonstrating regain of protein secretion. Upon treatment with the M3 muscarinic agonist acetylcholine, acini-like structures activated the fluid production pathway by increasing intracellular calcium levels. The increase in intracellular calcium seen in structures in the 3D hydrogel culture system was more robust and prolonged than that in 2.5D. To compare the long-term survival and retention of acini-like structures in vivo, cell-seeded 2.5D and 3D hydrogels were implanted into an athymic rat model. Cells in 2.5D failed to maintain organized acini-like structures and dispersed in the surrounding tissue. Encapsulated cells in 3D retained their spheroid structure and structural integrity, along with the salivary biomarkers and maintained viability for over 3 weeks in vivo. This report identifies a novel hydrogel culture system capable of creating and maintaining functional 3D salivary spheroid structures for long periods in vitro that regain both fluid and protein secreting functions and are suitable for tissue restoration.
PMCID: PMC3665323  PMID: 23442148
14.  Adipose-Derived Stem Cells Promote Angiogenesis and Tissue Formation for In Vivo Tissue Engineering 
Tissue Engineering. Part A  2013;19(11-12):1327-1335.
Adult mesenchymal stem cells secrete a variety of angiogenic cytokines and growth factors, so we proposed that these paracrine mechanisms may be used to promote vascularization and growth for tissue engineering in vivo. We tested whether or not human adipose-derived stem cells (ASCs) promote tissue formation in rats. ASCs were evaluated in vitro for mRNA expression of angiogenic factors, including the vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 (IL-8), and stromal cell-derived factor-1 (SDF-1) and proliferative activity on human microvascular endothelial cells. For in vivo analysis, CM-DiI-labeled ASCs were implanted with a rat cardiac extracellular matrix (ECM) extract-derived hydrogel into a chamber with a femoral arteriovenous loop in the groin of male nude rats for 7 days. Vascularization in newly generated tissue was estimated by histomorphometry after endothelial nitric oxide synthase (eNOS) immunostaining. ASCs expressed growth factor mRNA and produced an angiogenic activity in vitro. After implantation, ASCs survived, but remained suspended in the ECM and relatively few were incorporated into the newly formed tissue. The volume of newly generated tissue was significantly higher in chambers containing ASCs and it was enriched with vasculature when compared with the ECM alone. We conclude that human ASCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue-engineering applications for regenerative therapy.
PMCID: PMC3638514  PMID: 23394225
15.  Modulating the Physical Microenvironment to Study Regenerative Processes In Vitro Using Cells from Mouse Phalangeal Elements 
Tissue Engineering. Part A  2013;19(11-12):1406-1415.
Epimorphic regeneration in humans of complex multitissue structures is primarily limited to the digit tip. In a comparable mouse model, the response is level-specific in that regeneration occurs after amputation at the distal end of the terminal phalanx, but not more proximally. Recent isolation of stromal cells from CD1 murine phalangeal elements two and three (P2 and P3) allow for comparative studies of cells prevalent at the amputation plane of a more proximal region (considered nonregenerative) and a more distal region (considered regenerative), respectively. This study used adherent, suspension, and collagen gel cultures to investigate cellular processes relevant to the initial response to injury. Overall, P2 cells were both more migratory and able to compact collagen gels to a greater extent compared to P3 cells. This observed increased capacity of P2 cells to generate traction forces was likely related to the higher expression of key cytoskeletal proteins (e.g., microfilament, nonkeratin intermediate filaments, and microtubules) compared to P3 cells. In contrast, P3 cells were found to be more proliferative than P2 cells under all three culture conditions and to have higher expression of keratin proteins. In addition, when cultured in suspension rather than on adherent surfaces, P3 cells were both more proliferative and had greater gene expression for matrix proteins. Together these results add to the known inherent differences in these stromal cells by characterizing responses to the physical microenvironment. Further, while compaction by P2 cells confirm that collagen gels is a useful model to study wound healing, the response of P3 cells indicate that suspension culture, in which cell–cell interactions dominate like in the blastema, may be better suited to study regeneration. Therefore, this study can help develop clinical strategies for promoting regeneration through increased understanding in the properties of cells involved in endogenous repair as well as informed selection of useful in vitro models.
PMCID: PMC3638516  PMID: 23327620
16.  Tailoring Adipose Stem Cell Trophic Factor Production with Differentiation Medium Components to Regenerate Chondral Defects 
Tissue Engineering. Part A  2013;19(11-12):1451-1464.
Recent endeavors to use stem cells as trophic factor production sources have the potential to translate into viable therapies for damaged or diseased musculoskeletal tissues. Adipose stem cells (ASCs) can be differentiated into chondrocytes using the chondrogenic medium (CM), but it is unknown if this approach can optimize ASC growth factor secretion for cartilage regeneration by increasing the chondrogenic factor production, while decreasing angiogenic and hypertrophic factor production. The objective of this study was to determine the effects the CM and its components have on growth factor production from ASCs to promote cartilage regeneration. ASCs isolated from male Sprague-Dawley rats and cultured in monolayer or alginate microbeads were treated with either the growth medium (GM) or the CM for 5 days. In subsequent studies, ASC monolayers were treated with either the GM supplemented with different combinations of 50 μg/mL ascorbic acid-2-phosphate (AA2P), 100 nM dexamethasone (Dex), 10 ng/mL transforming growth factor (TGF)-β1, and 100 ng/mL bone morphogenetic protein (BMP)-6 or with the CM excluding different combinations of AA2P, Dex, TGF-β1, and BMP-6. mRNA levels and growth factor production were quantified at 8 and 24 h after the last media change, respectively. The CM increased chondrogenic factor secretion (TGF-β2, TGF-β3, and insulin-like growth factor [IGF]-I) and decreased angiogenic factor production (the vascular endothelial growth factor [VEGF]-A, the fibroblast growth factor [FGF]-2). Microencapsulation in the GM increased production of the chondrogenic (IGF-I, TGF-β2) and angiogenic (VEGF-A) factors. AA2P increased secretion of chondrogenic factors (IGF-I, TGF-β2), and decreased angiogenic factor (VEGF-A) secretion, in addition to decreasing mRNA levels for factors associated with chondrocyte hypertrophy (FGF-18). Dex increased mRNA levels for hypertrophic factors (BMP-2, FGF-18) and decreased angiogenic factor secretion (VEGF-A). TGF-β1 increased angiogenic factor production (FGF-2, VEGF-A) and decreased chondrogenic factor mRNA levels (IGF-I, PTHrP). BMP-6 increased hypertrophic mRNA levels (FGF-18) and chondrogenic factor production (TGF-β2). When ASC microbeads preconditioned with the CM were implanted in a focal cartilage defect and immobilized within an RGD-conjugated hydrogel, tissue infiltration from the edges of the defect and perichondrium was observed. These results show that differentiation media components have distinct effects on ASC's production of angiogenic, chondrogenic, and hypertrophic factors and that AA2P may be the most beneficial CM component for preconditioning ASCs to stimulate cartilage regeneration.
PMCID: PMC3638517  PMID: 23350662
17.  PLG Scaffold Delivered Antigen-Specific Regulatory T Cells Induce Systemic Tolerance in Autoimmune Diabetes 
Tissue Engineering. Part A  2013;19(11-12):1465-1475.
Islet transplantation is a promising treatment for human type 1 diabetes mellitus. Transplantation requires systemic immunosuppression, which has numerous deleterious side effects. Islet antigen-specific regulatory T cells (Tregs) have been shown to protect islet grafts from autoimmune destruction in the nonobese diabetic (NOD) model when co-localized in the kidney capsule. An extra-hepatic transplant site was established by transplanting islet-loaded microporous poly (lactide-co-glycolide) (PLG) scaffolds into abdominal fat. This study examined an autoimmune transplantation model and determined whether co-localized Tregs could protect islet grafts in an extra-hepatic and extra-renal transplant site. Normoglycemia was restored, and co-transplanted Tregs extended graft survival, including several instances of indefinite protection. Transplanted Tregs were replaced by recipient-derived Tregs over time, indicating that islet antigen-specific Tregs induce tolerance to islet grafts through host-derived Tregs. Thus, Tregs provided protection against a diverse repertoire of autoreactive T-cell-receptor specificities mediating diabetes in the NOD model, possibly through a phenomenon previously described as infectious tolerance. Interestingly, the infiltration by Tregs protected a second islet transplant, indicating systemic tolerance to islet antigens. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that allows for the co-localization of immunomodulatory cells within islet grafts and induces long-term graft survival in an autoimmune diabetes model. This method of co-localizing immunomodulatory cells with islets in a clinically translatable transplant site to affect the immune system on a local and systemic level has potential therapeutic implications for human islet transplantation.
PMCID: PMC3638535  PMID: 23432371
18.  Photofunctionalization of Alginate Hydrogels to Promote Adhesion and Proliferation of Human Mesenchymal Stem Cells 
Tissue Engineering. Part A  2013;19(11-12):1424-1432.
Photocrosslinkable biomaterials are promising for biomedical applications, as they can be injected in a minimally invasive manner, crosslinked in situ to form hydrogels with cells and/or bioactive factors, and engineered to provide instructive signals to transplanted and host cells. Our group has previously reported on biodegradable, photocrosslinkable alginate (ALG) hydrogels with controlled cell adhesivity for tissue engineering. The polymer backbone of this methacrylated ALG was covalently modified with cell adhesion ligands containing the RGD sequence to enhance the proliferation and differentiation response of encapsulated cells. However, this approach permits limited control over the spatial presentation of these ligands within the three-dimensional hydrogel structure. Here we present a system that easily allows for spatial control of cell adhesion ligands within photocrosslinked ALG hydrogels. A cell adhesive peptide composed of the specific amino acid sequence Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) was covalently modified with acrylate moieties. The acrylated peptide was then covalently incorporated into bulk hydrogels by adding it to methacrylated ALG solutions with a photoinitiator, and then photocrosslinking under long-wave ultraviolet light. The hydrogels were characterized with respect to their swelling and degradation profiles, and the effects of the acrylated peptide on human mesenchymal stem cell (hMSC) viability, adhesion, spreading, and proliferation were examined in vitro. hMSC adhesion and spreading on and proliferation in this biomaterial system could be regulated by varying the concentration of cell adhesion ligand. This new biomaterial system may be a useful platform for tissue engineering, drug delivery, and stem cell transplantation with spatial control of cell adhesivity.
PMCID: PMC3638536  PMID: 23327676
19.  Nonsteady State Oxygen Transport in Engineered Tissue: Implications for Design 
Tissue Engineering. Part A  2013;19(11-12):1433-1442.
Engineered tissue constructs are limited in size, and thus clinical relevance, when diffusion is the primary mode of oxygen transport. Understanding the extent of oxygen diffusion and cellular consumption is necessary for the design of engineered tissues, particularly those intended for implantation into hypoxic wound sites. This study presents a combined experimental and computation model to predict design constraints for cellularized fibrin tissues subjected to a step change in the oxygen concentration to simulate transplantation. Nonsteady state analysis of oxygen diffusion and consumption was used to estimate the diffusion coefficient of oxygen (mean±SD, 1.7×10−9±8.4×10−11 m2/s) in fibrin hydrogels as well as the Michaelis-Menten parameters, Vmax (1.3×10−17±9.2×10−19 mol·cell−1·s−1), and Km (8.0×10−3±3.5×0−3 mol/m3), of normal human lung fibroblasts. Nondimensionalization of the governing diffusion-reaction equation enabled the creation of a single dimensionless parameter, the Thiele modulus (φ), which encompasses the combined effects of oxygen diffusion, consumption, and tissue dimensions. Tissue thickness is the design parameter with the most pronounced influence on the distribution of oxygen within the system. Additionally, tissues designed such that φ<1 achieve a near spatially uniform and adequate oxygen concentration following the step change. Understanding and optimizing the Thiele modulus will improve the design of engineered tissue implants.
PMCID: PMC3638538  PMID: 23350630
20.  Stiffness-Modulated Water Retention and Neovascularization of Dermal Fibroblast-Encapsulating Collagen Gel 
Tissue Engineering. Part A  2013;19(11-12):1275-1284.
There is increasing evidence that matrix stiffness modulates various phenotypic activities of cells surrounded by a three-dimensional (3D) matrix. These findings suggest that matrix stiffness can also regulate dermal fibroblasts activities to remodel, repair, and recreate skin dermis, but this has not yet been systematically demonstrated to date. This study examines the effects of matrix rigidity on the morphology, growth rates, and glycosaminoglycan (GAG) production of dermal fibroblasts cultured in collagen-based hydrogels with controlled elastic moduli. The elastic moduli (E) of collagen hydrogels were increased from 0.7 to 1.6 and 2.2 kPa by chemically cross-linking collagen fibrils with poly(ethylene glycol) disuccinimidylester. Increasing E of the hydrogel led to decreases in cellular spreading, nuclear aspect ratio, and growth rate. In contrast, the cellular GAG production level was elevated by increasing E from 0.7 to 1.6 kPa. The larger accumulation of GAG in the stiffer hydrogel led to increased water retention during exposure to air, as confirmed with magnetic resonance imaging. Additionally, in a chicken chorioallantoic membrane, a cell-encapsulating hydrogel with E of 1.6 kPa created dermis-like tissue with larger amount of GAG and density of blood vessels, while a cell–hydrogel construct with E of 0.7 kPa generated scar-like tissue. Overall, the results of this study will be highly useful for designing advanced tissue engineering scaffolds that can enhance the quality of a wide array of regenerated tissues including skin.
PMCID: PMC3638539  PMID: 23289622
21.  Incorporation of Aggrecan in Interpenetrating Network Hydrogels to Improve Cellular Performance for Cartilage Tissue Engineering 
Tissue Engineering. Part A  2013;19(11-12):1349-1359.
Interpenetrating network (IPN) hydrogels were recently introduced to the cartilage tissue engineering literature, with the approach of encapsulating cells in thermally gelling agarose that is then soaked in a poly(ethylene glycol) diacrylate (PEGDA) solution, which is then photopolymerized. These IPNs possess significantly enhanced mechanical performance desirable for cartilage regeneration, potentially allowing patients to return to weight-bearing activities quickly after surgical implantation. In an effort to improve cell viability and performance, inspiration was drawn from previous studies that have elicited positive chondrogenic responses to aggrecan, the proteoglycan largely responsible for the compressive stiffness of cartilage. Aggrecan was incorporated into the IPNs in conservative concentrations (40 μg/mL), and its effect was contrasted with the incorporation of chondroitin sulfate (CS), the primary glycosaminoglycan associated with aggrecan. Aggrecan was incorporated by physical entrapment within agarose and methacrylated CS was incorporated by copolymerization with PEGDA. The IPNs incorporating aggrecan or CS exhibited over 50% viability with encapsulated chondrocytes after 6 weeks. Both aggrecan and CS improved cell viability by 15.6% and 20%, respectively, relative to pure IPNs at 6 weeks culture time. In summary, we have introduced the novel approach of including a raw material from cartilage, namely aggrecan, to serve as a bioactive signal to cells encapsulated in IPN hydrogels for cartilage tissue engineering, which led to improved performance of encapsulated chondrocytes.
PMCID: PMC3638541  PMID: 23379843
22.  Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein 
Tissue Engineering. Part A  2013;19(11-12):1386-1397.
An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34−, and CD45−), which surround capillaries and microvessels, and (2) adventitial cells (CD146−, CD34+, and CD45−), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×105 cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration.
PMCID: PMC3638559  PMID: 23406369
23.  Cord Lining-Mesenchymal Stem Cells Graft Supplemented with an Omental Flap Induces Myocardial Revascularization and Ameliorates Cardiac Dysfunction in a Rat Model of Chronic Ischemic Heart Failure 
Tissue Engineering. Part A  2013;19(11-12):1303-1315.
Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium. The graft consisted of a mixture of 2×106 CL-MSC-GFP-Fluc and fibrin. Myocardial infarction (MI) was induced in nude rats and following confirmation of ensued heart failure with echocardiography 2 weeks after injury, therapeutic intervention was performed as follows: untreated (MI, n=7), CL-MSC graft (CL-MSCG, n=8), CL-MSCG and omental flap (CL-MSCG+OM, n=11), and omental flap (OM, n=8). In vivo bioluminescence imaging at 1, 3, 7, and 14 days post-treatment indicated comparable early donor cell viability between the CL-MSCG and CL-MSCG+OM. Treatment with CL-MSCG+OM improved the myocardial function as assessed by the measurement of end-diastolic left ventricular (LV) pressure (3.53±0.34 vs. 5.21±0.54 mmHg, p<0.05), contractility (+dP/dt, 3383.8±250.78 mmHg vs. 2464.9±191.8 mmHg, p<0.05), and the relaxation rate (−dP/dt, −2707.2±250.7 mmHg vs. 1948.7±207.8 mmHg, p<0.05), compared to MI control 6 weeks after ischemic injury. Furthermore, evidence of a 20.32% increase in the ejection fraction was observed in CL-MSCG+OM rats from week 2 to 6 after injury. Both CL-MSCG and CL-MSCG+OM led to an enhanced cardiac output (p<0.05), and attenuated the infarct size (35.7%±4.2% and 34.7%±4.8%), as compared to MI (60.7%±3.1%; p<0.01 and p<0.001, respectively). All treated groups had a higher arteriole density than controls. Yet, a higher amount of functional blood vessels, and a 20-fold increase in arteriole numbers were found in CL-MSCG+OM. Altogether, CL-MSCGs supplemented with vascular supply have the potential to repair the failing, chronically ischemic heart by improving myocardial revascularization, attenuating remodeling, and ameliorating cardiac dysfunction.
PMCID: PMC3638562  PMID: 23448654
24.  Stage-Specific Embryonic Antigen-4 Is Not a Marker for Chondrogenic and Osteogenic Potential in Cultured Chondrocytes and Mesenchymal Progenitor Cells 
Tissue Engineering. Part A  2013;19(11-12):1316-1326.
One important challenge for regenerative medicine is to produce a clinically relevant number of cells with consistent tissue-forming potential. Isolation and expansion of cells from skeletal tissues results in a heterogeneous population of cells with variable regenerative potential. A more consistent tissue formation could be achieved by identification and selection of potent progenitors based on cell surface molecules. In this study, we assessed the expression of stage-specific embryonic antigen-4 (SSEA-4), a classic marker of undifferentiated stem cells, and other surface markers in human articular chondrocytes (hACs), osteoblasts, and bone marrow-derived mesenchymal stromal cells (bmMSCs) and characterized their differentiation potential. Further, we sorted SSEA-4-expressing hACs and followed their potential to proliferate and to form cartilage in vitro. Cells isolated from cartilage and bone exhibited remarkably heterogeneous SSEA-4 expression profiles in expansion cultures. SSEA-4 expression levels increased up to ∼5 population doublings, but decreased following further expansion and differentiation cultures; levels were not related to the proliferation state of the cells. Although SSEA-4-sorted chondrocytes showed a slightly better chondrogenic potential than their SSEA-4-negative counterparts, differences were insufficient to establish a link between SSEA-4 expression and chondrogenic potential. SSEA-4 levels in bmMSCs also did not correlate to the cells' chondrogenic and osteogenic potential in vitro. SSEA-4 is clearly expressed by subpopulations of proliferating somatic cells with a MSC-like phenotype. However, the predictive value of SSEA-4 as a specific marker of superior differentiation capacity in progenitor cell populations from adult human tissue and even its usefulness as a stem cell marker appears questionable.
PMCID: PMC3638563  PMID: 23301556
25.  Attachment of Flexible Heparin Chains to Gelatin Scaffolds Improves Endothelial Cell Infiltration 
Tissue Engineering. Part A  2013;19(11-12):1336-1348.
Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vascularization of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans.
PMCID: PMC3638622  PMID: 23327585

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