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1.  Bioengineered bugs, drugs and contentious issues in patenting 
Bioengineered Bugs  2010;1(1):2-8.
Bioengineered bugs, as is the scope of this journal, have great potential in various practical applications. A corollary to bringing useful products to the market is that such products need protection from copying by other people or businesses. Such government-sponsored protections are legally enforced through a patent, copyright or trademark/trade secret system commonly known as intellectual property rights. A condition for obtaining a patent is that the invention must not be disclosed to public either through seminars, informal public disclosures or publications in journals, although in the United States, there is a one year grace period that is allowed to obtain a patent after public disclosure. This article describes my personal experience in obtaining a patent in 1980 on a genetically manipulated bacterium designed for oil spill cleanup. This patent application went through a series of court cases that finally ended up in the Supreme Court of the United States. I also mention a similar contentious legal issue that is on the horizon and that the readers of Bioengineered Bugs should be aware of. Finally, I have taken the opportunity to describe my current efforts to bring to the market some unique potential multi-disease-targeting candidate drugs from Pseudomonas aeruginosa and gonococci/meningococci that, if found non-toxic and efficacious in humans, will revolutionize the drug industry. To ensure their marketability, we are trying to develop a patent portfolio that will ensure that they will be legally protected and such protections will be broad-based and enforceable.
doi:10.4161/bbug.1.1.9850
PMCID: PMC3035147  PMID: 21327122
anticancer drugs; business methods; drug promiscuity; HIV/AIDS; life form; malaria; patenting; Pseudomonas aeruginosa
2.  Functional Tooth Regeneration Using a Bioengineered Tooth Unit as a Mature Organ Replacement Regenerative Therapy 
PLoS ONE  2011;6(7):e21531.
Donor organ transplantation is currently an essential therapeutic approach to the replacement of a dysfunctional organ as a result of disease, injury or aging in vivo. Recent progress in the area of regenerative therapy has the potential to lead to bioengineered mature organ replacement in the future. In this proof of concept study, we here report a further development in this regard in which a bioengineered tooth unit comprising mature tooth, periodontal ligament and alveolar bone, was successfully transplanted into a properly-sized bony hole in the alveolar bone through bone integration by recipient bone remodeling in a murine transplantation model system. The bioengineered tooth unit restored enough the alveolar bone in a vertical direction into an extensive bone defect of murine lower jaw. Engrafted bioengineered tooth displayed physiological tooth functions such as mastication, periodontal ligament function for bone remodeling and responsiveness to noxious stimulations. This study thus represents a substantial advance and demonstrates the real potential for bioengineered mature organ replacement as a next generation regenerative therapy.
doi:10.1371/journal.pone.0021531
PMCID: PMC3134195  PMID: 21765896
3.  Welcome to Journal of Foot and Ankle Research: a new open access journal for foot health professionals 
Journal of Foot and Ankle Research (JFAR) is a new, open access, peer-reviewed online journal that encompasses all aspects of policy, organisation, delivery and clinical practice related to the assessment, diagnosis, prevention and management of foot and ankle disorders. JFAR will cover a wide range of clinical subject areas, including diabetology, paediatrics, sports medicine, gerontology and geriatrics, foot surgery, physical therapy, dermatology, wound management, radiology, biomechanics and bioengineering, orthotics and prosthetics, as well the broad areas of epidemiology, policy, organisation and delivery of services related to foot and ankle care. The journal encourages submission from all health professionals who manage lower limb conditions, including podiatrists, nurses, physical therapists and physiotherapists, orthopaedists, manual therapists, medical specialists and general medical practitioners, as well as health service researchers concerned with foot and ankle care. All manuscripts will undergo open peer review, and all accepted manuscripts will be freely available on-line using the open access platform of BioMed Central.
doi:10.1186/1757-1146-1-1
PMCID: PMC2547890  PMID: 18822156
4.  Bioengineered Dental Tissues Grown in the Rat Jaw 
Journal of dental research  2008;87(8):745-750.
Our long-term objective is to develop methods to form, in the jaw, bioengineered replacement teeth that exhibit physical properties and functions similar to those of natural teeth. Our results show that cultured rat tooth bud cells, seeded onto biodegradable scaffolds, implanted into the jaws of adult rat hosts and grown for 12 weeks, formed small, organized, bioengineered tooth crowns, containing dentin, enamel, pulp, and periodontal ligament tissues, similar to identical cell-seeded scaffolds implanted and grown in the omentum. Radiographic, histological, and immunohistochemical analyses showed that bioengineered teeth consisted of organized dentin, enamel, and pulp tissues. This study advances practical applications for dental tissue engineering by demonstrating that bioengineered tooth tissues can be regenerated at the site of previously lost teeth, and supports the use of tissue engineering strategies in humans, to regenerate previously lost and/or missing teeth. The results presented in this report support the feasibility of bioengineered replacement tooth formation in the jaw.
PMCID: PMC3024580  PMID: 18650546
tooth tissue engineering; dental stem cells; mandibular model
5.  Dynamic, Nondestructive Imaging of a Bioengineered Vascular Graft Endothelium 
PLoS ONE  2013;8(4):e61275.
Bioengineering of vascular grafts holds great potential to address the shortcomings associated with autologous and conventional synthetic vascular grafts used for small diameter grafting procedures. Lumen endothelialization of bioengineered vascular grafts is essential to provide an antithrombogenic graft surface to ensure long-term patency after implantation. Conventional methods used to assess endothelialization in vitro typically involve periodic harvesting of the graft for histological sectioning and staining of the lumen. Endpoint testing methods such as these are effective but do not provide real-time information of endothelial cells in their intact microenvironment, rather only a single time point measurement of endothelium development. Therefore, nondestructive methods are needed to provide dynamic information of graft endothelialization and endothelium maturation in vitro. To address this need, we have developed a nondestructive fiber optic based (FOB) imaging method that is capable of dynamic assessment of graft endothelialization without disturbing the graft housed in a bioreactor. In this study we demonstrate the capability of the FOB imaging method to quantify electrospun vascular graft endothelialization, EC detachment, and apoptosis in a nondestructive manner. The electrospun scaffold fiber diameter of the graft lumen was systematically varied and the FOB imaging system was used to noninvasively quantify the affect of topography on graft endothelialization over a 7-day period. Additionally, results demonstrated that the FOB imaging method had a greater imaging penetration depth than that of two-photon microscopy. This imaging method is a powerful tool to optimize vascular grafts and bioreactor conditions in vitro, and can be further adapted to monitor endothelium maturation and response to fluid flow bioreactor preconditioning.
doi:10.1371/journal.pone.0061275
PMCID: PMC3621659  PMID: 23585885
6.  Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens 
PLoS ONE  2012;7(10):e46884.
Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.
doi:10.1371/journal.pone.0046884
PMCID: PMC3466204  PMID: 23056510
7.  Phenotypic Characterization of Prostate Cancer LNCaP Cells Cultured within a Bioengineered Microenvironment 
PLoS ONE  2012;7(9):e40217.
Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.
doi:10.1371/journal.pone.0040217
PMCID: PMC3434144  PMID: 22957009
8.  Bioengineered 3D Human Kidney Tissue, a Platform for the Determination of Nephrotoxicity 
PLoS ONE  2013;8(3):e59219.
The staggering cost of bringing a drug to market coupled with the extremely high failure rate of prospective compounds in early phase clinical trials due to unexpected human toxicity makes it imperative that more relevant human models be developed to better predict drug toxicity. Drug–induced nephrotoxicity remains especially difficult to predict in both pre-clinical and clinical settings and is often undetected until patient hospitalization. Current pre-clinical methods of determining renal toxicity include 2D cell cultures and animal models, both of which are incapable of fully recapitulating the in vivo human response to drugs, contributing to the high failure rate upon clinical trials. We have bioengineered a 3D kidney tissue model using immortalized human renal cortical epithelial cells with kidney functions similar to that found in vivo. These 3D tissues were compared to 2D cells in terms of both acute (3 days) and chronic (2 weeks) toxicity induced by Cisplatin, Gentamicin, and Doxorubicin using both traditional LDH secretion and the pre-clinical biomarkers Kim-1 and NGAL as assessments of toxicity. The 3D tissues were more sensitive to drug-induced toxicity and, unlike the 2D cells, were capable of being used to monitor chronic toxicity due to repeat dosing. The inclusion of this tissue model in drug testing prior to the initiation of phase I clinical trials would allow for better prediction of the nephrotoxic effects of new drugs.
doi:10.1371/journal.pone.0059219
PMCID: PMC3597621  PMID: 23516613
9.  Production of a Bioengineered G-Protein Coupled Receptor of Human Formyl Peptide Receptor 3 
PLoS ONE  2011;6(8):e23076.
G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.
doi:10.1371/journal.pone.0023076
PMCID: PMC3154916  PMID: 21853070
10.  Study of Bioengineered Zebra Fish Olfactory Receptor 131-2: Receptor Purification and Secondary Structure Analysis 
PLoS ONE  2010;5(11):e15027.
How fishes are able to detect trace molecules in large bodies of water is not understood. It is plausible that they use olfactory receptors to detect water-soluble compounds. How the zebra fish Danio Rerio, an organism with only 98 functional olfactory receptors, is able to selectively detect and recognize numerous compounds in water remains a puzzling phenomenon. We are interested in studying the biochemical and molecular mechanisms of olfaction in fish. Here, we report on the study of a bioengineered zebra fish olfactory receptor OR131-2, affinity-purified from a HEK293S tetracycline-inducible system. This receptor was expressed and translocated to the cell plasma membrane as revealed by confocal microscopy. Circular dichroism spectroscopy showed that the purified zebra fish receptor folded into an α-helical structure, as observed for other G-protein coupled receptors (GPCRs). Our study shows that it is possible to produce viable quantities of the zebra fish olfactory receptor. This will not only enable detailed structural and functional analyses, but also aid in the design of biosensor devices in order to detect water-soluble metabolites or its intermediates, which are associated with human health.
doi:10.1371/journal.pone.0015027
PMCID: PMC2993934  PMID: 21124770
11.  Synergistic Actions of Hematopoietic and Mesenchymal Stem/Progenitor Cells in Vascularizing Bioengineered Tissues 
PLoS ONE  2008;3(12):e3922.
Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34+ hematopoietic cells. Co-transplantation of CD34+ HSCs and CD34− MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34+ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34+ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34+ cells. Based on additional in vitro results of endothelial differentiation of CD34+ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34+ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34+ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.
doi:10.1371/journal.pone.0003922
PMCID: PMC2597748  PMID: 19081793
12.  Bioengineered Human and Allogeneic Pulmonary Valve Conduits Chronically Implanted Orthotopically in Baboons: Hemodynamic Performance and Immunological Consequences 
Objective
This study assesses in a baboon model the hemodynamics and HLA immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning treatments) human and baboon heart valve scaffolds.
Methods
Fourteen baboons underwent pulmonary valve replacement, eight with decellularized and conditioned (bioengineered) pulmonary valves derived from either allogeneic (N=3) or xenogeneic (human) (N=5) hearts; for comparison, six baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel reactive serum antibodies (HLA Class I&II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms (TEE) measured valve function and geometry. Differences were analyzed with Kruskal-Wallis and Wilcoxon Rank Sum. P≤ 0.05 significant.
Results
All animals survived and thrived, exhibiting excellent immediate implanted valve function by TEE. Over time, reference valves developed smaller indexed effective orifice areas, EOAI=0.84(1.22) cm2/m2 median (range) while all bioengineered valves remained normal, EOAI=2.45 (1.35) cm2/m2; P=0.005. None of the bioengineered valves developed elevated peak transvalvular gradients, 5.5(6.0) versus 12.5(23.0) mmHg, P=0.003. Cryopreserved valves provoked the most intense antibody responses. Two of five human bioengineered and two of three baboon bioengineered valves did not provoke any Class I antibodies. Bioengineered human (but not baboon) scaffolds provoked Class II antibodies. C1q+ antibodies developed in four recipients.
Conclusions
Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves.
doi:10.1016/j.jtcvs.2012.06.024
PMCID: PMC3530005  PMID: 22841171
valves; surgery; xeno-transplantation; bioengineering; cardiac valve prostheses; tissue engineering; homografts; allografts; pulmonary valve
13.  Purification and cytotoxcicity of tag-free bioengineered spider silk proteins3 
Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in E. coli. The proteins were free of tag-sequence and were purified using two methods based on (i) thermal and (ii) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and since the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications.
doi:10.1002/jbm.a.34353
PMCID: PMC3494781  PMID: 22865581
spider silk; bioengineering; tag-free purification; cytotoxicity; immunogenicity
14.  Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop 
Summary
Background
Heterologous expression of Factor VIII (FVIII) is about 2 to 3 orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels.
Objective
Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study.
Methods and Results
Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS-1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2-fold. Finally, the addition of the A1-domain mutation, F309S in conjunction with the disulfide mutation had an additive effect resulting in a net improvement in secretion of between 35–45 fold higher than wild type FVIII in CHO cells.
Conclusion
Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII toward low cost factor replacement therapy for hemophilia A.
doi:10.1111/j.1538-7836.2011.04545.x
PMCID: PMC3290727  PMID: 22044596
bioengineering; disulfide bonds; factor VIII; hemophilia A; secretion
15.  Bioengineering and the cardiovascular system 
The development of the modern era of bioengineering and the advances in our understanding of the cardiovascular system have been intertwined over the past one-half century. This is true of bioengineering as an area for research in universities. Bioengineering is ultimately the beginning of a new engineering discipline, as well as a new discipline in the medical device industry.
doi:10.5339/gcsp.2013.5
PMCID: PMC3963728
16.  Bioengineered Periodontal Tissue Formed on Titanium Dental Implants 
Journal of Dental Research  2011;90(2):251-256.
The ability to use autologous dental progenitor cells (DPCs) to form organized periodontal tissues on titanium implants would be a significant improvement over current implant therapies. Based on prior experimental results, we hypothesized that rat periodontal ligament (PDL)-derived DPCs can be used to bioengineer PDL tissues on titanium implants in a novel, in vivo rat maxillary molar implant model. Analyses of recovered implants revealed organized PDL tissues surrounding titanium implant surfaces in PDL-cell-seeded, and not in unseeded control, implants. Rat PDL DPCs also exhibited differentiative potential characteristic of stem cells. These proof-of-principle findings suggest that PDL DPCs can organize periodontal tissues in the jaw, at the site of previously lost teeth, indicating that this method holds potential as an alternative approach to osseointegrated dental implants. Further refinement of this approach will facilitate the development of clinically relevant methods for autologous PDL regeneration on titanium implants in humans.
doi:10.1177/0022034510384872
PMCID: PMC3144101  PMID: 21149858
periodontal ligament; bioengineered tissues; titanium; dental implants
17.  Visualization of Morphological and Molecular Features Associated with Chronic Ischemia in Bioengineered Human Skin 
We present an in vitro model of human skin that, together with nonlinear optical microscopy, provides a useful system for characterizing morphological and structural changes in a living skin tissue microenvironment due to changes in oxygen status and proteolytic balance. We describe for the first time the effects of chronic oxygen deprivation on a bioengineered model of human interfollicular epidermis. Histological analysis and multiphoton imaging revealed a progressively degenerating ballooning phenotype of the keratinocytes that manifested after 48 h of hypoxic exposure. Multiphoton images of the dermal compartment revealed a decrease in collagen structural order. Immunofluorescence analysis showed changes in matrix metalloproteinase (MMP)-2 protein spatial localization in the epidermis with a shift to the basal layer, and loss of Ki67 expression in proliferative basal cells after 192 h of hypoxic exposure. Upon reoxygenation MMP-2 mRNA levels showed a biphasic response, with restoration of MMP-2 levels and localization. These results indicate that chronic oxygen deprivation causes an overall degeneration in tissue architecture, combined with an imbalance in proteolytic expression and a decrease in proliferative capacity. We propose that these tissue changes are representative of the ischemic condition and that our experimental model system is appropriate for addressing mechanisms of susceptibility to chronic wounds.
doi:10.1017/S1431927610000103
PMCID: PMC3249977  PMID: 20199713
hypoxia; ischemia; ulceration; bioengineered skin; multiphoton; MMP-2
18.  Liver bioengineering: Current status and future perspectives 
The present review aims to illustrate the strategies that are being implemented to regenerate or bioengineer livers for clinical purposes. There are two general pathways to liver bioengineering and regeneration. The first consists of creating a supporting scaffold, either synthetically or by decellularization of human or animal organs, and seeding cells on the scaffold, where they will mature either in bioreactors or in vivo. This strategy seems to offer the quickest route to clinical translation, as demonstrated by the development of liver organoids from rodent livers which were repopulated with organ specific cells of animal and/or human origin. Liver bioengineering has potential for transplantation and for toxicity testing during preclinical drug development. The second possibility is to induce liver regeneration of dead or resected tissue by manipulating cell pathways. In fact, it is well known that the liver has peculiar regenerative potential which allows hepatocyte hyperplasia after amputation of liver volume. Infusion of autologous bone marrow cells, which aids in liver regeneration, into patients was shown to be safe and to improve their clinical condition, but the specific cells responsible for liver regeneration have not yet been determined and the underlying mechanisms remain largely unknown. A complete understanding of the cell pathways and dynamics and of the functioning of liver stem cell niche is necessary for the clinical translation of regenerative medicine strategies. As well, it will be crucial to elucidate the mechanisms through which cells interact with the extracellular matrix, and how this latter supports and drives cell fate.
doi:10.3748/wjg.v18.i47.6926
PMCID: PMC3531676  PMID: 23322990
Liver; Regenerative medicine; Tissue engineering; Extracellular matrix; Scaffold; Stem cells
19.  Adult Human Gingival Epithelial Cells as a Source for Whole-tooth Bioengineering 
Journal of Dental Research  2013;92(4):329-334.
Teeth develop from interactions between embryonic oral epithelium and neural-crest-derived mesenchyme. These cells can be separated into single-cell populations and recombined to form normal teeth, providing a basis for bioengineering new teeth if suitable, non-embryonic cell sources can be identified. We show here that cells can be isolated from adult human gingival tissue that can be expanded in vitro and, when combined with mouse embryonic tooth mesenchyme cells, form teeth. Teeth with developing roots can be produced from this cell combination following transplantation into renal capsules. These bioengineered teeth contain dentin and enamel with ameloblast-like cells and rests of Malassez of human origin.
doi:10.1177/0022034513481041
PMCID: PMC3706174  PMID: 23458883
biotooth; tissue engineering; root formation; oral epithelium; ameloblast
20.  Improved targeting and enhanced retention of the human, autologous, fibroblast-derived, induced, pluripotent stem cells to the sarcomeres of the infarcted myocardium with the aid of the bioengineered, heterospecific, tetravalent antibodies** 
Clinical trials, to regenerate the human heart injured by myocardial infarction, involve the delivery of stem cells to the site of the injury. However, only a small fraction of the introduced stem cells are detected at the site of the injury, merely two weeks after this therapeutic intervention. This significantly hampers the effectiveness of the stem cell therapy.
To resolve the aforementioned problem, we genetically and molecularly bioengineered heterospecific, tetravalent antibodies (htAbs), which have both exquisite specificity and high affinity towards human, pluripotent, stem cells through the htAbs’ domains binding SSEA-4, SSEA-3, TRA-1-60, and TRA-1-81, as well as towards the injured cardiac muscle through the htAbs’ domains binding human cardiac myosin, α-actinin, actin, and titin. The cardiac tissue was acquired from the patients, who were receiving heart transplants. The autologous, human, induced, pluripotent stem cells (hiPSCs) were generated from the patients’ fibroblasts by non-viral delivery and transient expression of the DNA constructs for: Oct4, Nanog, Sox2, Lin28, Klf4, c-Myc. In the trials involving the htAbs, the human, induced, pluripotent stem cells anchored to the myocardial sarcomeres with the efficiency, statistically, significantly higher, than in the trials with non-specific or without antibodies (p < 0.0003). Moreover, application of the htAbs resulted in cross-linking of the sarcomeric proteins to create the stable scaffolds for anchoring of the stem cells. Thereafter, these human, induced pluripotent stem cells differentiated into cardiomyocytes at their anchorage sites.
By bioengineering of these novel heterospecific, tetravalent antibodies and using them to guide and to anchor the stem cells specifically to the stabilized sarcomeric scaffolds, we demonstrated the proof of concept in vitro for improving effectiveness of regenerative therapy of myocardial infarction and created the foundations for the trials in vivo.
PMCID: PMC3743128  PMID: 23956947
Myocardial infarction; heart attack; ischemic heart disease; stem cell therapy; cardiac regeneration; myofibril; SSEA-4; SSEA-3; TRA-1-60; TRA-1-81; myosin; α-actinin; actin; titin; desmin; troponin; bi-specific antibody; heterospecific tetravalent antibody; tetrabody
21.  Bioengineering Human Microvascular Networks in Immunodeficient Mice 
The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.
doi:10.3791/3065
PMCID: PMC3196173  PMID: 21775960
22.  Surgical implantation of a bioengineered internal anal sphincter☆ 
Journal of pediatric surgery  2010;45(1):52-58.
Purpose
Fecal incontinence is a common disorder that can have devastating social and psychologic consequences. However, there are no long-term ideal solutions for such patients. Although loss of continence is multifactorial, the integrity of the internal anal sphincter (IAS) has particular significance. We previously described the development of 3-dimensional bioengineered constructs using isolated smooth muscle tissue from donor C57BL/6 IAS. We hypothesized that the bioengineered ring constructs would retain cellular viability and promote neovascularization upon implantation into a recipient mouse.
Methods
Internal anal sphincter ring constructs were surgically implanted into the subcutaneous tissue of syngeneic C57BL/6 mice and treated with either fibroblastic growth factor 2 (0.26 µg daily) or saline controls using a microosmotic pump. Internal anal sphincter constructs were harvested after 25 days (range, 23–26 days) and assessed morphologically and for tissue viability.
Result
Gross morphology showed that there was no rejection. Rings showed muscle attachment to the back of the mouse with no sign of inflammation. Fibroblastic growth factor 2 infusion resulted in a significantly improved histologic score and muscle viability compared with the control group.
Conclusions
Three-dimensional bioengineered IAS rings can be successfully implanted into the subcutaneous tissue of recipient mice. The addition of fibroblastic growth factor 2 led to improved muscle viability, vascularity, and survival. This approach may become a feasible option for patients with fecal incontinence.
doi:10.1016/j.jpedsurg.2009.10.010
PMCID: PMC3018766  PMID: 20105579
Internal anal sphincter; Fibroblastic growth factor 2; Incontinence; Tissue engineering
23.  Bioengineering in the oral cavity: our experience 
Background
To date, there are no studies reported in the literature on the possible use of bovine collagen, oxidized regenerated cellulose, or synthetic hyaluronic acid medications in the oral cavity. The aim of this paper is to report the use of bovine collagen, oxidized regenerated cellulose, and synthetic hyaluronic acid medications to improve wound healing in the oral cavity by stimulating granulomatous tissue.
Methods
From 2007 to 2011, 80 patients (median age 67 years) suffering from oral mucosal lesions participated in this double-blind study. The patients were divided into two groups, each consisting of 40 patients. One group received conventional medications, while the other group of patients were treated with the advanced medications.
Results
Advanced medications allowed re-epithelialization of the wound margin in 2–20 days, whereas patients receiving conventional medication showed a median healing duration of 45 days.
Conclusion
The results of this study demonstrate that treating oral mucosal wounds with advanced medication has an advantage with regard to wound healing time, allowing patients to have a rapid, functional, and esthetic recovery.
doi:10.2147/IJN.S47697
PMCID: PMC3797608  PMID: 24143092
bioengineering; oral cavity; mucosal recovery
24.  Response surface optimization of the heparosan N-deacetylation in producing bioengineered heparin 
Journal of biotechnology  2011;156(3):188-196.
The chemical step in the chemoenzymatic synthesis of bioengineered heparin has been examined and optimized statistically using a response surface methodology. A four factor, two level full factorial design experiment and a three factor Box-Behnken design were carried out. The goal was to establish a method to prepare N-sulfo, N-acetyl heparosan of the desired N-acetyl content, number average molecular weight, and in maximum yield by controlling the reactant concentrations, reaction time and reaction temperature. The response surface models obtained were used to predict the reaction conditions required to optimally prepare N-sulfo, N-acetyl heparosan from E. coli generated heparosan starting material of different molecular weights.
doi:10.1016/j.jbiotec.2011.08.013
PMCID: PMC3196266  PMID: 21925548
25.  Shape, Loading, and Motion in the Bioengineering Design, Fabrication, and Testing of Personalized Synovial Joints 
Journal of biomechanics  2009;43(1):156.
With continued development and improvement of tissue engineering therapies for small articular lesions, increased attention is being focused on the challenge of engineering partial or whole synovial joints. Joint-scale constructs could have applications in the treatment of large areas of articular damage or in biological arthroplasty of severely degenerate joints. This review considers the roles of shape, loading and motion in synovial joint mechanobiology and their incorporation into the design, fabrication, and testing of engineered partial or whole joints. Incidence of degeneration, degree of impairment, and efficacy of current treatments are critical factors in choosing a target for joint bioengineering. The form and function of native joints may guide the design of engineered joint-scale constructs with respect to size, shape, and maturity. Fabrication challenges for joint-scale engineering include controlling chemo-mechano-biological microenvironments to promote the development and growth of multiple tissues with integrated interfaces or lubricated surfaces into anatomical shapes, and joint-scale bioreactors which nurture and stimulate the tissue with loading and motion. Finally, evaluation of load-bearing and tribological properties can range from tissue to joint scale and can focus on biological structure at present or after adaptation.
doi:10.1016/j.jbiomech.2009.09.021
PMCID: PMC2813363  PMID: 19815214
synovial joint; articular cartilage; tissue engineering; shape; motion

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