Publications by authors named "In Kap Ko"

38 Publications

Accelerating neovascularization and kidney tissue formation with a 3D vascular scaffold capturing native vascular structure.

Acta Biomater 2021 04 30;124:233-243. Epub 2021 Jan 30.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, NC, USA. Electronic address:

Establishing an adequate vascularization of three-dimensional (3D) bioengineered tissues remains a critical challenge. We previously fabricated a vascular scaffold using the vascular corrosion casting technique, which provides a similar 3D geometry of native kidney vasculature. In this study, we functionalized the collagen vascular scaffold with a controlled release of vascular endothelial growth factor (VEGF vascular scaffold) to further promote vascularization. The VEGF vascular scaffold showed improved angiogenic capability in 2-dimensional (2D) and 3D in vitro settings. Implantation of the VEGF vascular scaffold seeded with human renal cells into a rat kidney demonstrated enhanced implant vascularization and reduced apoptosis of implanted human renal cells. Hybrid renal tubule-like structures composed of implanted human and migrated host renal cells were formed. This work highlights the critical role of early vascularization of the geometrically mimetic vascular scaffold using the VEGF incorporated vascular scaffold in reducing apoptosis of implanted cells as well as the formation of renal tissue structures.
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http://dx.doi.org/10.1016/j.actbio.2021.01.040DOI Listing
April 2021

Pelvic floor muscle function recovery using biofabricated tissue constructs with neuromuscular junctions.

Acta Biomater 2021 02 13;121:237-249. Epub 2020 Dec 13.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1094, USA. Electronic address:

Damages in pelvic floor muscles often cause dysfunction of the entire pelvic urogenital system, which is clinically challenging. A bioengineered skeletal muscle construct that mimics structural and functional characteristics of native skeletal muscle could provide a therapeutic option to restore normal muscle function. However, most of the current bioengineered muscle constructs are unable to provide timely innervation necessary for successful grafting and functional recovery. We previously have demonstrated that post-synaptic acetylcholine receptors (AChR) clusters can be pre-formed on cultured skeletal muscle myofibers with agrin treatment and suggested that implantation of AChR clusters containing myofibers could accelerate innervation and recovery of muscle function. In this study, we develop a 3-dimensional (3D) bioprinted human skeletal muscle construct, consisting of multi-layers bundles with aligned and AChR clusters pre-formed human myofibers, and investigate the effect of pre-formed AChR clusters in bioprinted skeletal muscle constructs and innervation efficiency in vivo. Agrin treatment successfully pre-formed functional AChR clusters on the bioprinted muscle constructs in vitro that increased neuromuscular junction (NMJ) formation in vivo in a transposed nerve implantation model in rats. In a rat model of pelvic floor muscle injury, implantation of skeletal muscle constructs containing the pre-formed AChR clusters resulted in functional muscle reconstruction with accelerated construct innervation. This approach may provide a therapeutic solution to the many challenges associated with pelvic floor reconstruction resulting from the lack of suitable bioengineered tissue for efficient innervation and muscle function restoration.
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http://dx.doi.org/10.1016/j.actbio.2020.12.012DOI Listing
February 2021

Neural cell integration into 3D bioprinted skeletal muscle constructs accelerates restoration of muscle function.

Nat Commun 2020 02 24;11(1):1025. Epub 2020 Feb 24.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, 27157, USA.

A bioengineered skeletal muscle construct that mimics structural and functional characteristics of native skeletal muscle is a promising therapeutic option to treat extensive muscle defect injuries. We previously showed that bioprinted human skeletal muscle constructs were able to form multi-layered bundles with aligned myofibers. In this study, we investigate the effects of neural cell integration into the bioprinted skeletal muscle construct to accelerate functional muscle regeneration in vivo. Neural input into this bioprinted skeletal muscle construct shows the improvement of myofiber formation, long-term survival, and neuromuscular junction formation in vitro. More importantly, the bioprinted constructs with neural cell integration facilitate rapid innervation and mature into organized muscle tissue that restores normal muscle weight and function in a rodent model of muscle defect injury. These results suggest that the 3D bioprinted human neural-skeletal muscle constructs can be rapidly integrated with the host neural network, resulting in accelerated muscle function restoration.
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http://dx.doi.org/10.1038/s41467-020-14930-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039897PMC
February 2020

Reno-protection of Urine-derived Stem Cells in A Chronic Kidney Disease Rat Model Induced by Renal Ischemia and Nephrotoxicity.

Int J Biol Sci 2020 1;16(3):435-446. Epub 2020 Jan 1.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, USA.

Drug-induced nephrotoxicity can occur in patients with pre-existing renal dysfunction or renal ischemia, potentially leading to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Prompt treatment of CKD and the related side effects is critical in preventing progression to ESRD. The goal of this study was to demonstrate the therapeutic potential of urine-derived stem cells (USC) to treat chronic kidney disease-induced by nephrotoxic drugs and renal ischemia. Human USC were collected, expanded and characterized by flow cytometry. A CKD model was induced by creating an ischemia-reperfusion injury and gentamicin administration. Twenty-eight adult immunodeficient rats were divided into three groups: PBS-treated group (n=9), USC-treated group (n=9), and sham group with age-matched control animals (n=10). Cell suspension of USC (5 x 10 / 100µl / kidney) or PBS was injected bilaterally into the renal parenchyma 9 weeks after CKD model creation. Renal function was evaluated by collection blood and urine samples to measure serum creatinine and glomerulus filtration rate. The kidneys were harvested 12 weeks after cell injection. Histologically, the extent of glomerulosclerosis and tubular atrophy, the amount of collagen deposition, interstitial fibrosis, inflammatory monocyte infiltration, and expression of transforming growth factor beta 1 (TGF-ß1), and superoxide dismutase 1 (SOD-1) were examined. USC expressed renal parietal epithelial cells (CD24, CD29 and CD44). Renal function, measured by GFR and serum Cr in USC-treated group were significantly improved compared to PBS-treated animals (p<0.05). The degree of glomerular sclerosis and atrophic renal tubules, the amount of fibrosis, and monocyte infiltration significantly decreased in USC-treated group compared to the PBS group (p<0.05). The level of TGF-ß1 expression in renal tissues was also significantly lower in the PBS group, while the level of SOD-1 expression was significantly elevated in the USC group, compared to PBS group (p<0.05). The present study demonstrates the nephron-protective effect of USC on renal function via anti-inflammatory, anti-oxidative stress, and anti-fibrotic activity in a dual-injury CKD rat model. This provides an alternative treatment for CKD in certain clinical situations, such as instances where CKD is due to drug-induced nephrotoxicity and renal ischemia.
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http://dx.doi.org/10.7150/ijbs.37550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990904PMC
January 2021

State-of-the-Art Strategies for the Vascularization of Three-Dimensional Engineered Organs.

Vasc Specialist Int 2019 Jun 30;35(2):77-89. Epub 2019 Jun 30.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA.

Engineering three-dimensional (3D) implantable tissue constructs is a promising strategy for replacing damaged or diseased tissues and organs with functional replacements. However, the efficient vascularization of new 3D organs is a major scientific and technical challenge since large tissue constructs or organs require a constant blood supply to survive in vivo. Current approaches to solving this problem generally fall into the following three major categories: (a) cell-based, (b) angiogenic factor-based, and (c) scaffold-based. In this review, we summarize state-of-the-art technologies that are used to develop complex, stable, and functional vasculature for engineered 3D tissue constructs and organs; additionally, we have suggested directions for future research.
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http://dx.doi.org/10.5758/vsi.2019.35.2.77DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609020PMC
June 2019

Controlled Delivery of Stem Cell-Derived Trophic Factors Accelerates Kidney Repair After Renal Ischemia-Reperfusion Injury in Rats.

Stem Cells Transl Med 2019 09 30;8(9):959-970. Epub 2019 May 30.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina, USA.

Renal disease is a worldwide health issue. Besides transplantation, current therapies revolve around dialysis, which only delays disease progression but cannot replace other renal functions, such as synthesizing erythropoietin. To address these limitations, cell-based approaches have been proposed to restore damaged kidneys as an alternative to current therapies. Recent studies have shown that stem cell-derived secretomes can enhance tissue regeneration. However, many growth factors undergo rapid degradation when they are injected into the body in a soluble form. Efficient delivery and controlled release of secreting factors at the sites of injury would improve the efficacy in tissue regeneration. Herein, we developed a gel-based delivery system for controlled delivery of trophic factors in the conditioned medium (CM) secreted from human placental stem cells (HPSCs) and evaluated the effect of trophic factors on renal regeneration. CM treatment significantly enhanced cell proliferation and survival in vitro. Platelet-rich plasma (PRP) was used as a delivery vehicle for CM. Analysis of the release kinetics demonstrated that CM delivery through the PRP gel resulted in a controlled release of the factors both in vitro and in vivo. In an acute kidney injury model in rats, functional and structural analysis showed that CM delivery using the PRP gel system into the injured kidney minimized renal tissue damage, leading to a more rapid functional recovery when compared with saline, CM, or vehicle only injection groups. These results suggest that controlled delivery of HPSC-derived trophic factors may provide efficient repair of renal tissue injury. Stem Cells Translational Medicine 2019;8:959&970.
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http://dx.doi.org/10.1002/sctm.18-0222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708069PMC
September 2019

Use of uniformly sized muscle fiber fragments for restoration of muscle tissue function.

J Tissue Eng Regen Med 2019 07 29;13(7):1230-1240. Epub 2019 May 29.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC.

Treatment of extensive muscle loss due to traumatic injury, congenital defects, or tumor ablations is clinically challenging. The current treatment standard is grafting of autologous muscle flaps; however, significant donor site morbidity and graft tissue availability remain a problem. Alternatively, muscle fiber therapy has been attempted to treat muscle injury by transplanting single fibers into the defect site. However, irregularly organized long fibers resulted in low survivability due to delay in vascular and neural integration, thus limiting the therapeutic efficacy. Therefore, no effective method is available to permanently restore extensive muscle injuries. To address the current limitations, we developed a novel method that produces uniformly sized native muscle fiber fragments (MFFs) for muscle transplantation. We hypothesized that fragmentation of muscle fibers into small and uniformly sized fragments would allow for rapid reassembly and efficient engraftment within the defect site, resulting in accelerated recovery of muscle function. Our results demonstrate that the processed MFFs have a dimension of approximately 100 μm and contain living muscle cells on extracellular matrices. In preclinical animal studies using volumetric defect and urinary incontinence models, histological and functional analyses confirmed that the transplanted MFFs into the injury sites were able to effectively integrate with host muscle tissue, vascular, and neural systems, which resulted in significant improvement of muscle function and mass. These results indicate that the MFF technology platform is a promising therapeutic option for the restoration of muscle function and can be applied to various muscle defect and injury cases.
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http://dx.doi.org/10.1002/term.2873DOI Listing
July 2019

Kidney regeneration with biomimetic vascular scaffolds based on vascular corrosion casts.

Acta Biomater 2019 09 3;95:328-336. Epub 2019 Apr 3.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, NC, USA. Electronic address:

We have developed a biomimetic renal vascular scaffold based on a vascular corrosion casting technique. This study evaluated the feasibility of using this novel biomimetic scaffold for kidney regeneration in a rat kidney cortical defect model. Vascular corrosion casts were prepared from normal rat kidneys by perfusion with 10% polycaprolactone (PCL) solution, followed by tissue digestion. The corrosion PCL cast was coated with collagen, and PCL was removed from within the collagen coating, leaving only a hollow collagen-based biomimetic vascular scaffold. The fabricated scaffolds were pre-vascularized with MS1 endothelial cell coating, incorporated into 3D renal constructs, and subsequently implanted either with or without human renal cells in the renal cortex of nude rats. The implanted collagen-based vascular scaffold was easily identified and integrated into native kidney tissue. The biomimetic vascular scaffold coated with endothelial cells (MS1) showed significantly enhanced vascularization, as compared to the uncoated scaffold and hydrogel only groups (P < 0.001). Along with the improved vascularization effects, the MS1-coated scaffolds showed a significant renal cell infiltration from the neighboring host tissue, as compared to the other groups (P < 0.05). Moreover, addition of human renal cells to the MS1-coated scaffold resulted in further enhancement of vascularization and tubular structure regeneration within the implanted constructs. The biomimetic collagen vascular scaffolds coated with endothelial cells are able to enhance vascularization and facilitate the formation of renal tubules after 14 days when combined with human renal cells. This study shows the feasibility of bioengineering vascularized functional renal tissues for kidney regeneration. STATEMENT OF SIGNIFICANCE: Vascularization is one of the major hurdles affecting the survival and integration of implanted three-dimensional tissue constructs in vivo. A novel, biomimetic, collagen-based vascular scaffold that is structurally identical to native kidney tissue was developed and tested. This biomimetic vascularized scaffold system facilitates the development of new vessels and renal cell viability in vivo when implanted in a partial renal defect. The use of this scaffold system could address the challenges associated with vascularization, and may be an ideal treatment strategy for partial augmentation of renal function in patients with chronic kidney disease.
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http://dx.doi.org/10.1016/j.actbio.2019.04.001DOI Listing
September 2019

Effect of Human Amniotic Fluid Stem Cells on Kidney Function in a Model of Chronic Kidney Disease.

Tissue Eng Part A 2019 11 14;25(21-22):1493-1503. Epub 2019 Jun 14.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina.

Kidney disease is a major medical problem globally. Chronic kidney disease (CKD) is a progressive loss of kidney function. It causes accumulation of waste and fluid in the body, eventually resulting in kidney failure as well as damaging other organs. Although dialysis and kidney transplantation have been used as primary treatments for renal disease, dialysis does not restore full renal function, and there is a shortage of donor kidneys for transplantation. Recent advances in cell-based therapies have offered a means to augment and restore renal function. Various types of cells have been tested to evaluate their therapeutic effects on injured kidneys. Among various types of cells, amniotic fluid stem cells (AFSCs) share advantages of both embryonic and adult stem cells, such as pluripotent activity, remarkable plasticity, and immunomodulatory effects, which may allow their future therapeutic use as an "off-the-shelf" cell source. AFSC presents advantages of both conventional pluripotent and adult stem cells, such as pluripotent activity, remarkable plasticity, and immunomodulatory effects. This study demonstrates that administration of human-derived AFSC facilitates functional and structural improvement in a rat model of CKD, and suggests that cell therapy with AFSC has potential as a therapeutic strategy to recover renal function in patients with CKD. Impact Statement Patients with chronic kidney disease (CKD) have limited treatment options, and renal transplantation is the only definitive treatment method that restores kidney function. However, challenges associated with transplantation, including donor organ shortage, rejection, and life-long immunosuppression, remain a problem. Recently, stem cell-based therapies have been proposed as an alternative approach to augment and restore renal function. In this study, we used human-derived amniotic fluid stem cells (AFSCs) to treat CKD in a rat model and demonstrated that AFSC treatment facilitated positive effects in terms of improvements of renal function.
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http://dx.doi.org/10.1089/ten.TEA.2018.0371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059005PMC
November 2019

Bioactive Compounds for the Treatment of Renal Disease.

Yonsei Med J 2018 Nov;59(9):1015-1025

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA.

Kidney diseases including acute kidney injury and chronic kidney disease are among the largest health issues worldwide. Dialysis and kidney transplantation can replace a significant portion of renal function, however these treatments still have limitations. To overcome these shortcomings, a variety of innovative efforts have been introduced, including cell-based therapies. During the past decades, advances have been made in the stem cell and developmental biology, and tissue engineering. As part of such efforts, studies on renal cell therapy and artificial kidney developments have been conducted, and multiple therapeutic interventions have shown promise in the pre-clinical and clinical settings. More recently, therapeutic cell-secreting secretomes have emerged as a potential alternative to cell-based approaches. This approach involves the use of renotropic factors, such as growth factors and cytokines, that are produced by cells and these factors have shown effectiveness in facilitating kidney function recovery. This review focuses on the renotropic functions of bioactive compounds that provide protective and regenerative effects for kidney tissue repair, based on the available data in the literature.
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http://dx.doi.org/10.3349/ymj.2018.59.9.1015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192891PMC
November 2018

3D Bioprinted Human Skeletal Muscle Constructs for Muscle Function Restoration.

Sci Rep 2018 08 17;8(1):12307. Epub 2018 Aug 17.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, 27157, United States.

A bioengineered skeletal muscle tissue as an alternative for autologous tissue flaps, which mimics the structural and functional characteristics of the native tissue, is needed for reconstructive surgery. Rapid progress in the cell-based tissue engineering principle has enabled in vitro creation of cellularized muscle-like constructs; however, the current fabrication methods are still limited to build a three-dimensional (3D) muscle construct with a highly viable, organized cellular structure with the potential for a future human trial. Here, we applied 3D bioprinting strategy to fabricate an implantable, bioengineered skeletal muscle tissue composed of human primary muscle progenitor cells (hMPCs). The bioprinted skeletal muscle tissue showed a highly organized multi-layered muscle bundle made by viable, densely packed, and aligned myofiber-like structures. Our in vivo study presented that the bioprinted muscle constructs reached 82% of functional recovery in a rodent model of tibialis anterior (TA) muscle defect at 8 weeks of post-implantation. In addition, histological and immunohistological examinations indicated that the bioprinted muscle constructs were well integrated with host vascular and neural networks. We demonstrated the potential of the use of the 3D bioprinted skeletal muscle with a spatially organized structure that can reconstruct the extensive muscle defects.
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http://dx.doi.org/10.1038/s41598-018-29968-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098064PMC
August 2018

Comparative analysis of two porcine kidney decellularization methods for maintenance of functional vascular architectures.

Acta Biomater 2018 07 5;75:226-234. Epub 2018 Jun 5.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.

Kidney transplantation is currently the only definitive solution for the treatment of end-stage renal disease (ESRD), however transplantation is severely limited by the shortage of available donor kidneys. Recent progress in whole organ engineering based on decellularization/recellularization techniques has enabled pre-clinical in vivo studies using small animal models; however, these in vivo studies have been limited to short-term assessments. We previously developed a decellularization system that effectively removes cellular components from porcine kidneys. While functional re-endothelialization on the porcine whole kidney scaffold was able to improve vascular patency, as compared to the kidney scaffold only, the duration of patency lasted only a few hours. In this study, we hypothesized that significant damage in the microvasculatures within the kidney scaffold resulted in the cessation of blood flow, and that thorough investigation is necessary to accurately evaluate the vascular integrity of the kidney scaffolds. Two decellularization protocols [sodium dodecyl sulfate (SDS) with DNase (SDS + DNase) or Triton X-100 with SDS (TRX + SDS)] were used to evaluate and optimize the levels of vascular integrity within the kidney scaffold. Results from vascular analysis studies using vascular corrosion casting and angiograms demonstrated that the TRX + SDS method was able to better maintain intact and functional microvascular architectures such as glomeruli within the acellular matrices than that by the SDS + DNase treatment. Importantly, in vitro blood perfusion of the re-endothelialized kidney construct revealed improved vascular function of the scaffold by TRX + SDS treatment compared with the SDS + DNase. Our results suggest that the optimized TRX + SDS decellularization method preserves kidney-specific microvasculatures and may contribute to long-term vascular patency following implantation.

Statement Of Significance: Kidney transplantation is the only curative therapy for patients with end-stage renal disease (ESRD). However, in the United States, the supply of donor kidneys meets less than one-fifth of the demand; and those patients that receive a donor kidney need life-long immunosuppressive therapy to avoid organ rejection. In the last two decades, regenerative medicine and tissue engineering have emerged as an attractive alternative to overcome these limitations. In 2013, Song et al. published the first experimental orthotopic transplantation of a bioengineering kidney in rodents. In this study, they demonstrated evidences of kidney tissue regeneration and partial function restoration. Despite these initial promising results, there are still many challenges to achieve long-term blood perfusion without graft thrombosis. In this paper, we demonstrated that perfusion of detergents through the renal artery of porcine kidneys damages the glomeruli microarchitecture as well as peritubular capillaries. Modifying dynamic parameters such as flow rate, detergent concentration, and decellularization time, we were able to establish an optimized decellularization protocol with no evidences of disruption of glomeruli microarchitecture. As a proof of concept, we recellularized the kidney scaffolds with endothelial cells and in vitro perfused whole porcine blood successfully for 24 h with no evidences of thrombosis.
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http://dx.doi.org/10.1016/j.actbio.2018.06.004DOI Listing
July 2018

Bioartificial Kidneys.

Curr Stem Cell Rep 2017 Jun 12;3(2):68-76. Epub 2017 Apr 12.

Wake Forest Institute for Regenerative Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1083, USA.

Purpose Of Review: Historically, there have been many advances in the ways in which we treat kidney diseases. In particular, hemodialysis has set the standard for treatment since the early 1960s and continues today as the most common form of treatment for acute, chronic, and end-stage conditions. However, the rising global prevalence of kidney diseases and our limited understanding of their etiologies have placed significant burdens on current clinical management regimens. This has resulted in a desperate need to improve the ways in which we treat the underlying and ensuing causes of kidney diseases for those who are unable to receive transplants.

Recent Findings: One way of possibly addressing these issues is through the use of improved bioartificial kidneys. Bioartificial kidneys provide an extension to conventional artificial kidneys and dialysis systems, by incorporating aspects of living cellular and tissue function, in an attempt to better mimic normal kidneys. Recent advancements in genomic, cellular, and tissue engineering technologies are facilitating the improved design of these systems.

Summary: In this review, we outline various research efforts that have focused on the development of regenerated organs, implantable constructs, and whole bioengineered kidneys, as well as the transitions from conventional dialysis to these novel alternatives. As a result, we envision that these pioneering efforts can one day produce bioartificial renal technologies that can either perform or reintroduce essential function, and thus provide practical options to treat and potentially prevent kidney diseases.
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http://dx.doi.org/10.1007/s40778-017-0079-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526744PMC
June 2017

Progressive Muscle Cell Delivery as a Solution for Volumetric Muscle Defect Repair.

Sci Rep 2016 12 7;6:38754. Epub 2016 Dec 7.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.

Reconstructing functional volumetric tissue in vivo following implantation remains a critical challenge facing cell-based approaches. Several pre-vascularization approaches have been developed to increase cell viability following implantation. Structural and functional restoration was achieved in a preclinical rodent tissue defect; however, the approach used in this model fails to repair larger (>mm) defects as observed in a clinical setting. We propose an effective cell delivery system utilizing appropriate vascularization at the site of cell implantation that results in volumetric and functional tissue reconstruction. Our method of multiple cell injections in a progressive manner yielded improved cell survival and formed volumetric muscle tissues in an ectopic muscle site. In addition, this strategy supported the reconstruction of functional skeletal muscle tissue in a rodent volumetric muscle loss injury model. Results from our study suggest that our method may be used to repair volumetric tissue defects by overcoming diffusion limitations and facilitating adequate vascularization.
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http://dx.doi.org/10.1038/srep38754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141432PMC
December 2016

Induction of multiple ovulation via modulation of angiotensin II receptors in in vitro ovarian follicle culture models.

J Tissue Eng Regen Med 2017 11 2;11(11):3100-3110. Epub 2016 Nov 2.

Department of Obstetrics and Gynaecology, Seoul National University Hospital, Seoul, South Korea.

In vitro culture of ovarian follicles is a promising bioengineering technique for retrieving fertilizable oocytes from preserved ovarian tissues of cancer survivors. However, current in vitro follicle culture techniques are labour-intensive and of low efficiency, as only single follicle culture (SFC) has been possible to date. The present study investigated the feasibility of multifollicular cluster culture (MFCC) system using angiotensin II receptor (ATII-Rc) analogues. Ovarian pre-antral follicles isolated from 2-week-old C57BL6 mice were cultured with ATII-Rc agonist or antagonist and their maturation outcomes were compared with control group. When single follicles were cultured, the ovulation and maturation rates were similar in all three groups. When three-follicle clusters were cultured, up to three follicles were ovulated in the ATII-Rc agonist group while none or one follicle ovulated in control or antagonist groups (p < 0.0001). Significantly higher numbers of mature oocytes were obtained in the agonist group (three-follicle 28.2 ± 4.9 vs. SFC 11.0 ± 1.3, per 25 cultured droplets) (p < 0.0001), and the development of each fertilized oocytes was comparable to those from SFC. It is therefore concluded that this novel MFCC system can significantly improve the efficiency of in vitro mature oocyte retrieval via ATII-Rc modulation. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2214DOI Listing
November 2017

A 3D bioprinting system to produce human-scale tissue constructs with structural integrity.

Nat Biotechnol 2016 Mar 15;34(3):312-9. Epub 2016 Feb 15.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina, USA.

A challenge for tissue engineering is producing three-dimensional (3D), vascularized cellular constructs of clinically relevant size, shape and structural integrity. We present an integrated tissue-organ printer (ITOP) that can fabricate stable, human-scale tissue constructs of any shape. Mechanical stability is achieved by printing cell-laden hydrogels together with biodegradable polymers in integrated patterns and anchored on sacrificial hydrogels. The correct shape of the tissue construct is achieved by representing clinical imaging data as a computer model of the anatomical defect and translating the model into a program that controls the motions of the printer nozzles, which dispense cells to discrete locations. The incorporation of microchannels into the tissue constructs facilitates diffusion of nutrients to printed cells, thereby overcoming the diffusion limit of 100-200 μm for cell survival in engineered tissues. We demonstrate capabilities of the ITOP by fabricating mandible and calvarial bone, cartilage and skeletal muscle. Future development of the ITOP is being directed to the production of tissues for human applications and to the building of more complex tissues and solid organs.
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http://dx.doi.org/10.1038/nbt.3413DOI Listing
March 2016

Fabrication of biomimetic vascular scaffolds for 3D tissue constructs using vascular corrosion casts.

Acta Biomater 2016 Mar 6;32:190-197. Epub 2016 Jan 6.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA. Electronic address:

Unlabelled: Vascularization is among the most pressing technical challenges facing tissue engineering of 3D organs. While small engineered constructs can rely solely on vascular infiltration and diffusion from host tissues following implantation, larger avascular constructs do not survive long enough for vessel ingrowth to occur. To address this challenge, strategies for pre-vascularization of engineered constructs have been developed. Various biofabrication techniques have been utilized for pre-vascularization, but limitations exist with respect to the size and complexity of the resulting vessels. To this end, we developed a simple and novel fabrication method to create biomimetic microvascular scaffolds using vascular corrosion casting as a template for pre-vascularization of engineered tissue constructs. Gross and electron microscopic analysis demonstrates that polycaprolactone (PCL)-derived kidney vascular corrosion casts are able to capture the architecture of normal renal tissue and can serve as a sacrificial template for the creation of a collagen-based vascular scaffold. Histological analysis demonstrates that the collagen vascular scaffolds are biomimetic in structure and can be perfused, endothelialized, and embedded in hydrogel tissue constructs. Our scaffold creation method is simple, cost effective, and provides a biomimetic, tissue-specific option for pre-vascularization that is broadly applicable in tissue engineering.

Statement Of Significance: Tissues in the body are vascularized to provide nutrients to the cells within the tissues and carry away waste, but creating tissue engineered constructs with functional vascular networks has been challenging. Current biofabrication techniques can incorporate blood vessel-like structures with straight or simple branching patterns into tissue constructs. Unfortunately, these techniques are expensive, complicated and create simplified versions of the complex vessel structures seen in native tissue. Our technique uses novel vascular corrosion casts of normal tissue as templates to create vascular scaffolds that are a copy of normal vessels. These vascular scaffolds can be easily incorporated into 3D tissue constructs. Our process is simple, inexpensive and inherently tissue-specific, making it widely applicable in the field of tissue engineering.
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http://dx.doi.org/10.1016/j.actbio.2016.01.005DOI Listing
March 2016

Repopulation of porcine kidney scaffold using porcine primary renal cells.

Acta Biomater 2016 Jan 17;29:52-61. Epub 2015 Nov 17.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.

Unlabelled: The only definitive treatment for end stage renal disease is renal transplantation, however the current shortage of organ donors has resulted in a long list of patients awaiting transplant. Whole organ engineering based on decellularization/recellularization techniques has provided the possibility of creating engineered kidney constructs as an alternative to donor organ transplantation. Previous studies have demonstrated that small units of engineered kidney are able to maintain function in vivo. However, an engineered kidney with sufficient functional capacity to replace normal renal function has not yet been developed. One obstacle in the generation of such an organ is the development of effective cell seeding methods for robust colonization of engineered kidney scaffolds. We have developed cell culture methods that allow primary porcine renal cells to be efficiently expanded while maintaining normal renal phenotype. We have also established an effective cell seeding method for the repopulation of acellular porcine renal scaffolds. Histological and immunohistochemical analyses demonstrate that a majority of the expanded cells are proximal tubular cells, and the seeded cells formed tubule-like structures that express normal renal tubule phenotypic markers. Functional analysis revealed that cells within the kidney construct demonstrated normal renal functions such as re-adsorption of sodium and protein, hydrolase activity, and production of erythropoietin. These structural and functional outcomes suggest that engineered kidney scaffolds may offer an alternative to donor organ transplant.

Statement Of Significance: Kidney transplantation is the only definitive treatment for end stage renal disease, however the current shortage of organ donors has limited the treatment. Whole organ engineering based on decellularization/recellularization techniques has provided the possibility of creating engineered kidney constructs as an alternative to donor organ transplantation. While previous studies have shown that small units of engineered kidneys are able to maintain function in animal studies, engineering of kidneys with sufficient functional capacity to replace normal renal function is still challenging due to inefficient cell seeding methods. This study aims to establish an effective cell seeding method using pig kidney cells for the repopulation of acellular porcine kidney scaffolds, suggesting that engineered kidneys may offer an alternative to donor organ transplant.
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http://dx.doi.org/10.1016/j.actbio.2015.11.026DOI Listing
January 2016

Kidney diseases and tissue engineering.

Methods 2016 Apr 29;99:112-9. Epub 2015 Jun 29.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA. Electronic address:

Kidney disease is a worldwide public health problem. Renal failure follows several disease stages including acute and chronic kidney symptoms. Acute kidney injury (AKI) may lead to chronic kidney disease (CKD), which can progress to end-stage renal disease (ESRD) with a mortality rate. Current treatment options are limited to dialysis and kidney transplantation; however, problems such as donor organ shortage, graft failure and numerous complications remain a concern. To address this issue, cell-based approaches using tissue engineering (TE) and regenerative medicine (RM) may provide attractive approaches to replace the damaged kidney cells with functional renal specific cells, leading to restoration of normal kidney functions. While development of renal tissue engineering is in a steady state due to the complex composition and highly regulated functionality of the kidney, cell therapy using stem cells and primary kidney cells has demonstrated promising therapeutic outcomes in terms of restoration of renal functions in AKI and CKD. In this review, basic components needed for successful renal kidney engineering are discussed, and recent TE and RM approaches to treatment of specific kidney diseases will be presented.
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http://dx.doi.org/10.1016/j.ymeth.2015.06.020DOI Listing
April 2016

Cell-based therapy for kidney disease.

Korean J Urol 2015 Jun 27;56(6):412-21. Epub 2015 May 27.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, USA.

The prevalence of renal disease continues to increase worldwide. When normal kidney is injured, the damaged renal tissue undergoes pathological and physiological events that lead to acute and chronic kidney diseases, which frequently progress to end stage renal failure. Current treatment of these renal pathologies includes dialysis, which is incapable of restoring full renal function. To address this issue, cell-based therapy has become a potential therapeutic option to treat renal pathologies. Recent development in cell therapy has demonstrated promising therapeutic outcomes, in terms of restoration of renal structure and function impaired by renal disease. This review focuses on the cell therapy approaches for the treatment of kidney diseases, including various cell sources used, as well recent advances made in preclinical and clinical studies.
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http://dx.doi.org/10.4111/kju.2015.56.6.412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462630PMC
June 2015

Whole kidney engineering for clinical translation.

Curr Opin Organ Transplant 2015 Apr;20(2):165-70

aWake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA bKonkuk University Medical College, Seoul, Korea.

Purpose Of Review: Renal transplantation is currently the only definitive treatment for end-stage renal disease; however, this treatment is severely limited by the shortage of implantable kidneys. To address this shortcoming, development of an engineered, transplantable kidney has been proposed. Although current advances in engineering kidneys based on decellularization and recellularization techniques have offered great promises for the generation of functional kidney constructs, most studies have been conducted using rodent kidney constructs and short-term in-vivo evaluation. Toward clinical translations of this technique, several limitations need to be addressed.

Recent Findings: Human-sized renal scaffolds are desirable for clinical application, and the fabrication is currently feasible using native porcine and discarded human kidneys. Current progress in stem cell biology and cell culture methods have demonstrated feasibility of the use of embryonic stem cells, induced pluripotent stem cells, and primary renal cells as clinically relevant cell sources for the recellularization of renal scaffolds. Finally, approaches to long-term implantation of engineered kidneys are under investigation using antithrombogenic strategies such as functional reendothelialization of acellular kidney matrices.

Summary: In the field of bioengineering, whole kidneys have taken a number of important initial steps toward clinical translations, but many challenges must be addressed to achieve a successful treatment for the patient with end-stage renal disease.
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http://dx.doi.org/10.1097/MOT.0000000000000173DOI Listing
April 2015

Bioengineered transplantable porcine livers with re-endothelialized vasculature.

Biomaterials 2015 Feb 26;40:72-9. Epub 2014 Nov 26.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.

Donor shortage remains a continued challenge in liver transplantation. Recent advances in tissue engineering have provided the possibility of creating functional liver tissues as an alternative to donor organ transplantation. Small bioengineered liver constructs have been developed, however a major challenge in achieving functional bioengineered liver in vivo is the establishment of a functional vasculature within the scaffolds. Our overall goal is to bioengineer intact livers, suitable for transplantation, using acellular porcine liver scaffolds. We developed an effective method for reestablishing the vascular network within decellularized liver scaffolds by conjugating anti-endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells. This procedure resulted in uniform endothelial attachment throughout the liver vasculature extending to the capillary bed of the liver scaffold and greatly reduced platelet adhesion upon blood perfusion in vitro. The re-endothelialized livers, when transplanted to recipient pigs, were able to withstand physiological blood flow and maintained for up to 24 h. This study demonstrates, for the first time, that vascularized bioengineered livers, of clinically relevant size, can be transplanted and maintained in vivo, and represents the first step towards generating engineered livers for transplantation to patients with end-stage liver failure.
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http://dx.doi.org/10.1016/j.biomaterials.2014.11.027DOI Listing
February 2015

In situ tissue regeneration through host stem cell recruitment.

Exp Mol Med 2013 Nov 15;45:e57. Epub 2013 Nov 15.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, USA.

The field of tissue engineering has made steady progress in translating various tissue applications. Although the classical tissue engineering strategy, which involves the use of culture-expanded cells and scaffolds to produce a tissue construct for implantation, has been validated, this approach involves extensive cell expansion steps, requiring a lot of time and laborious effort before implantation. To bypass this ex vivo process, a new approach has been introduced. In situ tissue regeneration utilizes the body's own regenerating capacity by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the site of injury. This approach relies on development of a target-specific biomaterial scaffolding system that can effectively control the host microenvironment and mobilize host stem/progenitor cells to target tissues. An appropriate microenvironment provided by implanted scaffolds would facilitate recruitment of host cells that can be guided to regenerating structural and functional tissues.
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http://dx.doi.org/10.1038/emm.2013.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849571PMC
November 2013

The effect of in vitro formation of acetylcholine receptor (AChR) clusters in engineered muscle fibers on subsequent innervation of constructs in vivo.

Biomaterials 2013 Apr 4;34(13):3246-55. Epub 2013 Feb 4.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA.

Timely innervation of muscle tissue is critical in the recovery of function, and this time-sensitive process relies heavily on the host tissue microenvironment after implantation. However, restoration of muscle tissue mass and function has been a challenge. We investigated whether pre-forming acetylcholine receptor (AChR) clusters on engineered muscle fibers using an AChR cluster-inducing factor (agrin) prior to implantation would facilitate established contacts between implanted muscle tissues and nerves and result in rapid innervation of engineered muscle in vivo. We showed that agrin treatment significantly increased the formation of AChR clusters on culture differentiated myotubes (C2C12), enhanced contacts with nerves in vitro and in vivo, and increased angiogenesis. Pre-fabrication of AChR clusters on engineered skeletal muscle using a released neurotrophic factor can accelerate innervations following implantation in vivo. This technique has considerable potential for enhancing muscle tissue function.
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http://dx.doi.org/10.1016/j.biomaterials.2013.01.029DOI Listing
April 2013

Amniotic fluid-derived stem cells in regenerative medicine research.

Arch Pharm Res 2012 Feb 28;35(2):271-80. Epub 2012 Feb 28.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.

The stem cells isolated from amniotic fluid present an exciting possible contribution to the field of regenerative medicine and amniotic fluid-derived stem (AFS) cells have significant potential for research and therapeutic applications. AFS cells are multipotent, showing the ability to differentiate into cell types from all three embryonic germ layers. They express both embryonic and adult stem cell markers, expand extensively without feeder cells, double in 36 h, and are not tumorigenic. The AFS cells can be maintained for over 250 population doublings and preserve their telomere length and a normal karyotype. They differentiate easily into specific cell lineages and do not require human embryo tissue for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem (ES) cells. The discovery of the AFS cells has been recent, and a great deal of work remains to be performed on the characterization and use of these cells. This review describes the various differentiated lineages that AFS cells can form and the future of these promising new stem cells in regenerative medicine research.
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http://dx.doi.org/10.1007/s12272-012-0207-7DOI Listing
February 2012

Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration.

FASEB J 2012 Jan 30;26(1):158-68. Epub 2011 Sep 30.

Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA.

Whereas the conventional tissue engineering strategy involves the use of scaffolds combined with appropriate cell types to restore normal functions, the concept of in situ tissue regeneration uses host responses to a target-specific scaffold to mobilize host cells to a site of injury without the need for cell seeding. For this purpose, local delivery of bioactive molecules from scaffolds has been generally used. However, this approach has limited stem cell recruitment into the implants. Thus, we developed a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor-1α (SDF-1α) from an implant. In this study, we examined whether this combined system would significantly enhance recruitment of host stem cells into the implants. Flow cytometry and immunohistochemistry for CD29/CD45, CD146/α-smooth muscle actin, and c-kit demonstrated that this system significantly increased the number of stem cell-like cells within the implants when compared with other systems. In vitro culture of the cells that had infiltrated into the scaffolds from the combined system confirmed that host stem cells were recruited into these implants and indicated that they were capable of differentiation into multiple lineages. These results indicate that this combined system may lead to more efficient tissue regeneration.
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http://dx.doi.org/10.1096/fj.11-182998DOI Listing
January 2012

Targeting improves MSC treatment of inflammatory bowel disease.

Mol Ther 2010 Jul 13;18(7):1365-72. Epub 2010 Apr 13.

Department of Orthopedics, Case Western Reserve University, Cleveland, Ohio, USA.

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is an inflammatory autoimmune disease characterized by T-cell infiltration to the colon. Mesenchymal stem cells (MSCs) have the potential to rescue IBD owing to their immunosuppressive capabilities and clinical studies have shown positive influence on intestinal graft versus host disease. We demonstrate here a new method to coat MSCs with antibodies against addressins to enhance their delivery to the colon and thereby increase the therapeutic effectiveness. Bioluminescence imaging (BLI) demonstrated that vascular cell adhesion molecule antibody (Ab)-coated MSCs (Ab(VCAM-1)- MSCs) had the highest delivery efficiency to inflamed mesenteric lymph node (MLN) and colon compared to untreated MSCs, Ab(isotype)-MSCs, and Ab(MAdCAM)-MSCs. Therapeutically, when mice with IBD were injected with addressin Ab-coated MSCs, they showed dramatically improved survival rates, higher IBD therapeutic scores, and significantly improved body weight gain compared to mice injected with MSCs only, isotype Ab, free Ab plus MSCs, or vehicle-only controls. These data demonstrate that anti-addressin Ab coating on MSC increased cell delivery to inflamed colon and increased the efficacy of MSC treatment of IBD. This is the first study showing an increased therapeutic efficacy when stem cells are first coated with antibodies specifically target them to inflamed sites.
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http://dx.doi.org/10.1038/mt.2010.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911249PMC
July 2010

Targeting mesenchymal stem cells to activated endothelial cells.

Biomaterials 2009 Aug 17;30(22):3702-10. Epub 2009 Apr 17.

Department of Orthopaedics, Case Western Reserve University, Cleveland, OH, USA.

Cell surface coating is a methodology wherein specific molecules are transiently anchored onto cell membrane to modulate cell behavior. Cell surface coating was tested as a method to deliver mesenchymal stem cells (MSCs) to endothelial cells via binding to intercellular cell adhesion molecule-1 (ICAM-1). MSCs coated with palmitated protein G (PPG) followed by antibodies to ICAM-1 (Ab(ICAM-1)), and incubated on ICAM-I coated coverslips showed a 40-fold increase in cell binding over PPG-only controls. Ab(ICAM-1)-coated MSCs incubated with human vascular endothelial cells (HUVECs), with and without exposure to TNFalpha (to upregulate ICAM-1 expression), showed 2.6-fold increased binding to control HUVECs over PPG-only controls, and a 16-fold increase in binding to TNFalpha-treated HUVECs. Pretreatment of HUVECs with ICAM-1 antibody promoted the attachment of PPG-only MSCs while reducing the attachment of Ab(ICAM-1)-MSCs by approximately 50%. In flow chamber studies on TNFalpha-stimulated HUVECs, PPG-only, and MSC-only lost 80-90% of their initial binding at 4 dyne/cm(2), while Ab(ICAM-1)-MSCs maintained 100% binding at 4 dyne/cm(2) and 40% binding at 25 dyne/cm(2). These results demonstrate that cell surface coating promotes the attachment of MSCs to endothelial cells, and provides a methodology for the delivery of stem cells to sites of inflammation.
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http://dx.doi.org/10.1016/j.biomaterials.2009.03.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693266PMC
August 2009

Acid-degradable cationic methacrylamide polymerized in the presence of plasmid DNA as tunable non-viral gene carrier.

Biomaterials 2008 Oct 27;29(28):3872-81. Epub 2008 Jun 27.

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA.

New acid-degradable cationic nanoparticles were synthesized using a monomer-to-polymer approach, which enabled highly flexible nanoparticle fabrication to obtain controlled properties such as size and conjugation with additional functionalities. The nanoparticles were designed to cause swelling and osmotic destabilization of the endosome, while cationic branches holding anionic DNA are cleaved from the polymeric backbone of the nanoparticles and make plasmid DNA accessible for efficient gene expression. Efficient release of plasmid DNA upon hydrolysis of the nanoparticles at the endosomal pH 5.0 and transportation of the released DNA to the nucleus of a cell were shown. In vitro studies showed significantly higher transfection efficiency by the degradable nanoparticles than polyethylenimine (PEI) polyplexes at very low concentrations (i.e., ng/mL). Size-dependent selective transfection of phagocytic cells (e.g., RAW 309 macrophages) and non-phagocytic cells (e.g., NIH 3T3 fibroblasts) was also achieved by using nanoparticles of two different sizes (240 nm and 680 nm in diameter), which implies feasibility of tunable gene therapy and DNA vaccination using the nanoparticle system. Preliminary pulmonary transfection of mice using the degradable nanoparticles demonstrated a remarkably higher expression of firefly luciferase at 70% lower concentration than using naked DNA alone. Implications and further improvement of the nanoparticles to be used in gene therapy are also discussed.
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http://dx.doi.org/10.1016/j.biomaterials.2008.06.003DOI Listing
October 2008

In vivo MR imaging of tissue-engineered human mesenchymal stem cells transplanted to mouse: a preliminary study.

Ann Biomed Eng 2007 Jan 3;35(1):101-8. Epub 2006 Nov 3.

Department of Radiology, College of Medicine, 134 ShinChon-Dong, Seodaemun-Ku, Seoul, 120-752, Republic of Korea.

Current progress integrating stem cell biology and tissue engineering techniques has been invaluable to clinical applications. Prior to the application of cellular transplantation technique to patients, we need to establish techniques that can monitor their tissue biodistribution non-invasively. In this study, we proposed an imaging modality using MRI to not only monitor implanted scaffold in vivo, but also to track transplanted cells and behavior around the implant. For this purpose, human bone marrow-derived mesenchymal stem cells (hMSCs) were labeled with superparamagnetic iron oxide (Feridex) and then labeled hMSCs were cultured in a gelatin sponge used as a scaffold to support cell growth and proliferation. Histological assessment and MTT assay showed that cell labeling with MR contrast agent did not harm cell viability. Also, Feridex-labeled hMSCs showed a significant decrease in T2 signal intensity, even within the gelatin sponge in vitro. After implanting the sponge/cell complex in vivo, we could visualize cellular behavior around the implant over time using a noninvasive MRI modality and this finding was correlated with histological study, which illustrates the potential of a new approach proposed here for in vivo monitoring of implanted cell-based tissue-engineered product.
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http://dx.doi.org/10.1007/s10439-006-9204-7DOI Listing
January 2007