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We report here on the development and characterization of a cell-based system for the regulated delivery of bioactive insulin-like growth factor I (IGF-I). A stable mammalian cell line, CHO-K1 Tet-IGFI, was genetically modified to have tetracycline-induced transcription of the human IGF-I gene. Cells were activated to express IGF-I in the presence of doxycycline (DOX), a tetracycline derivative, while expression was inactivated in the absence of DOX. Temporal, or on-off, release of IGF-I from cells encapsulated within Ca²⁺-alginate hydrogels was demonstrated in a pilot study over the course of 10 days in culture. Released growth factor was bioactive, exhibiting a proliferative effect comparable to recombinant purified IGF-I protein. The dosage levels and temporal control of IGF-I release from encapsulated cells meet the requirements of orthopedic wound repair, making this approach an attractive means for the controlled synthesis and delivery of growth factors in situ for wound healing.

This report draws upon data from a variety of sources to provide a detailed estimate of the current scope of private sector development and commercial activity in the aggregate field comprising tissue engineering, regenerative medicine, and stem cell therapeutics. Economic activity has grown a remarkable fivefold in the past 5 years. As of mid-2007 approximately 50 firms or business units with over 3000 employees offered commercial tissue-regenerative products or services with generally profitable annual sales in excess of $1.3 billion. Well over a million patients have been treated with these products. In addition, 110 development-stage companies with over 55 products in FDA-level clinical trials and other preclinical stages employed approximately 2500 scientists or support personnel and spent 850 million development dollars in 2007. These totals represent a remarkable recovery from the downturn of 2000-2002, at which time tissue engineering was in shambles because of disappointing product launches, failed regulatory trials, and the general investment pullback following the dot-com crash. Commercial success has resulted in large measure from identification of products that are achievable with available technology and under existing regulatory guidelines. Development-stage firms have become much more adept at risk management. The resilience of the field, as well as its current breadth and diversity, augurs well for the future of regenerative medicine.

The article summarizes the collective views expressed at the fourth session of the workshop Tissue Engineering--the Next Generation, which was devoted to the translation of results of tissue engineering research into applications. Ernst Hunziker described the paradigm of a dual translational approach, and argued that tissue engineering should be guided by the dimensions and physiological setting of the bodily compartment to be repaired. Myron Spector discussed collagen-glycosaminoglycan (GAG) scaffolds for musculoskeletal tissue engineering. Jeanette Libera focused on the biological and clinical aspects of cartilage tissue engineering, and described a completely autologous procedure for engineering cartilage using the patient's own chondrocytes and blood serum. Arthur Gertzman reviewed the applications of allograft tissues in orthopedic surgery, and outlined the potential of allograft tissues as models for biological and medical studies. Savio Woo discussed a list of functional tissue engineering approaches designed to restore the biochemical and biomechanical properties of injured ligaments and tendons to be closer to that of the normal tissues. Specific examples of using biological scaffolds that have chemoattractants as well as growth factors with unique contact guidance properties to improve their healing process were shown. Anthony Ratcliffe discussed the translation of the results of research into products that are profitable and meet regulatory requirements. Michael Lysaght challenged the proposition that commercial and clinical failures of early tissue engineering products demonstrate a need for more focus on basic research. Arthur Coury described the evolution of tissue engineering products based on the example of Genzyme, and how various definitions of success and failure can affect perceptions and policies relative to the status and advancement of the field of tissue engineering.

The source of insulin released from insulin-releasing cell clusters (IRCCs) differentiated from embryonic stem cells remains unclear. Rajagopal et al. have suggested that IRCCs do not synthesize but secrete insulin that had been absorbed from media during the multistep protocol. We report here further data relevant to this controversy. No radioisotopic labeling of insulin was observed when IRCCs were incubated in a medium containing 35S-cysteine. Less than 1% of the extra-cellular stoichiometric C-peptide equivalent to insulin was secreted during glucose stimulation. However, intracellular immunostaining and immunogold labeling were both positive for C-peptide. Finally, a mass balance calculation showed that simple equilibration of IRCCs by Fickian diffusion from media accounted for at most 4% of secreted insulin. These findings and further analysis of the results of others suggest that the mechanism of insulin secretion by IRCCs is a combination of sequestration and de novo synthesis.

This study was undertaken to assess the impact of current economic conditions and recent disappointing product launches on the field of tissue engineering. Data were collected on all firms known to be active in the field, analyzed, and compared with analogous data collected in 1995, 1998, and 2000. As of December 31, 2002, more than 2600 full-time equivalents (FTEs) in 15 countries and 89 firms were engaged in tissue-engineering research and development. Annual spending was US dollars 487 million, down about 20% since 2000-a reasonable performance in the face of a stagnant economy and difficult capital markets. Individual sectors proved far more volatile. Activity in skin, cartilage, and other structural applications declined by more than 50% with a loss of 800 FTEs. This downsizing was somewhat counterbalanced by a 42% increase in stem cell firms, which added more than 300 employees. Consistent with general disenchantment with technology sector equities, capital value of publicly traded tissue-engineering corporations has decreased by almost 90% from US dollars 2.5 billion at the end of 2000 to US dollars 300 million at the end of 2002. The United States' fraction of the total workforce declined from 80% in 2000 to 54% in 2002. By the close of 2002, twenty tissue-engineered products had entered Food and Drug Administration clinical trials. Four were approved but none of these are yet commercially successful. Six other applications were either abandoned or failed to achieve product approval. Ten products were still in clinical trials, some of which were investigator sponsored, and most of which were at the phase I/phase II stage. The field has yet to produce a profitable product despite an aggregate research and development investment exceeding US dollars 4.5 billion. Tissue engineering is clearly having difficulty transitioning from a development stage industry to one with a successful product portfolio. This is often the case for breakthrough medical technologies.

This report draws upon data from a variety of sources to estimate the size, scope, and growth rate of the contemporary tissue engineering enterprise. At the beginning of 2001, tissue engineering research and development was being pursued by 3,300 scientists and support staff in more than 70 startup companies or business units with a combined annual expenditure of over $600 million. Spending by tissue engineering firms has been growing at a compound annual rate of 16%, and the aggregate investment since 1990 now exceeds $3.5 billion. At the beginning of 2001, the net capital value of the 16 publicly traded tissue engineering startups had reached $2.6 billion. Firms focusing on structural applications (skin, cartilage, bone, cardiac prosthesis, and the like) comprise the fastest growing segment. In contrast, efforts in biohybrid organs and other metabolic applications have contracted over the past few years. The number of companies involved in stem cells and regenerative medicine is rapidly increasing, and this area represents the most likely nidus of future growth for tissue engineering. A notable recent trend has been the emergence of a strong commercial activity in tissue engineering outside the United States, with at least 16 European or Australian companies (22% of total) now active.

Effect of membrane composition and structure on solute removal and biocompatibility in hemodialysis. Significant changes in extracorporeal membranes have occurred over the past five decades in which hemodialysis (HD) has been available as a therapy for both acute renal failure (ARF) and end-stage renal disease (ESRD). For cellulosic membranes, these changes have included a reduction in thickness, hydroxyl group substitution, and an increase in pore size. These modifications have resulted in enhanced efficiency of small solute removal, a broader spectrum of overall solute removal, and an attenuation of complement activation in comparison to the thick, unsubstituted cellulosic membranes of low permeability used in the early days of HD therapy. Synthetic membranes, originally developed specifically for use in high-flux HD and hemofiltration, have also evolved during this same time period. In fact, the initially clear distinction between low-flux regenerated cellulosic and high-flux synthetic membranes has become blurred, as membrane formulators have developed products designed to appeal to enthusiasts for both membrane formats. The purpose of this review is to characterize both the solute removal and biocompatibility characteristics of dialysis membranes according to their composition (that is, polymeric makeup) and structure. In this regard, the manner in which membrane biocompatibility interacts with flux is highlighted.

The contemporary scope of worldwide tissue engineering research and development was estimated by totaling the relevant annual spending and other economic parameters of firms involved the field. Operating expenses allocated to tissue engineering in 1997 exceed $450 million and fund the activities of nearly 2,500 scientists and support personnel. Growth rate is 22.5% per annum. Most activity is centered in the United States. Government spending in this field represents10% of the total. The aggregate capital value of start-ups that have gone public was approximately $1.7 billion as of January 1, 1998; total capital value of all firms and business units in the field was estimated to be roughly $3.5 billion. The level of investment and valuation represents a remarkable act of faith in the future of a technology yet to produce its first significant revenue-generating product.

Immediately after the availability of highly permeable membranes in 1979, membrane plasma separation was introduced as a mode of extracorporeal blood purification by the nephrology group at Klinikum Grosshadern of the Ludwig Maximilians University of Munich (F.R.G.). The new therapy was applied primarily in the management of immunologically mediated renal and extrarenal disorders as well as in paraproteinemias. We also have witnessed a widespread application of this extracorporeal treatment as a last resort in otherwise refractory clinical conditions. Over the years, the group at Grosshadern has contributed to the development, as well as to the laboratory and clinical testing, of new plasma separation membranes, simplified plasmapheresis formats (e.g., spontaneous membrane plasma separation), and several plasma fractionation procedures (e.g., cascade filtration, adsorption). Whenever indicated and possible, plasma fractionation procedures, rather than unselective plasma exchange, are performed in an appropriate clinical situation.

Tissue engineering may be defined as the application of the principles and techniques of biomedical engineering to products and processes involving living cells. As such it is located at the confluence of traditional medical device technology, molecular pharmacology, and cell biology. Effective function at this interface has proven both elusive and critical in tissue engineering research and is likely to remain a determinant of success or failure as products move from research through development and into clinical practice. As a further challenge, regulatory processes, traditions, and approval pathways are rather different for medical devices than for Pharmaceuticals and biologies, and commercialization of hybrid products will require knowledge and expertise in both areas. This review compares and contrasts the development cycle for medical devices and pharmaceutical products and highlights the major development issues facing emerging tissue engineering products.

Artificial organs education is often an overlooked field for many bioengineering and biomedical engineering students. The purpose of this article is to describe three different approaches to teaching artificial organs. This article can serve as a reference for those who wish to offer a similar course at their own institutions or incorporate these ideas into existing courses. Artificial organ classes typically fulfill several ABET (Accreditation Board for Engineering and Technology) criteria, including those specific to bioengineering and biomedical engineering programs.

We report here on the development and characterization of a cell-based system for the regulated delivery of bioactive insulin-like growth factor I (IGF-I). A stable mammalian cell line, CHO-K1 Tet-IGFI, was genetically modified to have tetracycline-induced transcription of the human IGF-I gene. Cells were activated to express IGF-I in the presence of doxycycline (DOX), a tetracycline derivative, while expression was inactivated in the absence of DOX. Temporal, or on-off, release of IGF-I from cells encapsulated within Ca²⁺-alginate hydrogels was demonstrated in a pilot study over the course of 10 days in culture. Released growth factor was bioactive, exhibiting a proliferative effect comparable to recombinant purified IGF-I protein. The dosage levels and temporal control of IGF-I release from encapsulated cells meet the requirements of orthopedic wound repair, making this approach an attractive means for the controlled synthesis and delivery of growth factors in situ for wound healing.

Near-infrared (n.i.r.) spectroscopy in both the short wavelength region (700–1100 nm) and the long wavelength region (1100–1500 nm) is evaluated as a rapid method for the simultaneous estimation of the freezing point and volume percentages of aromatics and saturates of aviation fuels. Thirty-three JP-4 samples were independently analysed by six industrial and government laboratories for freezing point and for volume per cent aromatics and volume per cent saturates by fluorescent indicator adsorption. These results were then used to calibrate the sample's n.i.r. spectra. Both multiple linear regression and partial least squares calibration methods were evaluated and gave comparable performance. The n.i.r. spectral analysis predictions for these properties were within the standard deviations of the round-robin values. Specifically, the per cent aromatics and per cent saturates were within 1% and the freezing point within 2.5 °C of the laboratory-determined values. N.i.r. spectroscopy appears to have considerable promise as a rapid non-destructive method for jet fuel analysis.

Insulin-like growth factor 1 (IGF-1) is a potent mitogen and differentiation factor with particular relevance to orthopedic tissue engineering. A biologically based Ca2+-alginate microcapsule vehicle, utilizing genetically modified primary normal human fibroblasts (NHFs), was developed and characterized for localized synthesis and delivery of human IGF-1 (hIGF-1). Normal human fibroblasts were transfected to overexpress the hIGF-1 gene, leading to cells that expressed 4 ng of hIGF-1 per 10(6) cells per 24 hours. Encapsulation within alginate led to a six-fold enhancement in the generation and release of hIGF-1 to 22 ng of hIGF-1 per 10(6) cells per 24 hours. Release was constitutive, predictable, and exhibited highly repeatable first-order kinetics with no initial burst. Released growth factor was biologically active and exhibited a proliferative effect comparable to commercially available recombinant hIGF-1. The magnitude of hIGF-1 release met the requirements of orthopedic tissue generation, and this approach is considered an attractive alternative to other proposed methods of growth factor delivery.

We describe a fiber‐optic, field‐portable spectrophotometer for measurement in the near‐infrared (NIR) (680–1050 nm) region of the spectrum. This instrument, bundled with a laptop computer for data acquisition and statistical analysis, has a footprint of only 6 in.×14 in.×18 in. and weights approximately 20 pounds. The instrument uses the single beam measurement principle by employing a grating‐based spectrograph for wavelength dispersion and a silicon photodiode‐array (PDA) as detector. A computer‐controlled, optically stabilized 5.5‐W tungsten lamp is used as the light source. Sample measurements are made with a bifurcated fiber bundle for noninvasive diffuse reflectance and/or transflectance measurements. The portable spectrometer attains an RMS signal‐to‐noise ratio of 1.24×10−5 AU, stray light intensity levels of 0.1%, spectral resolution of 25 nm, and a dynamic range of approximately three orders of magnitude. The analysis of three‐component hydrocarbon mixtures by transflectance NIR spectroscopy is demonstrated.

Methods for calculating fluid and mass removal in peritoneal dialysis are presented in order to aid clinicians in their care and management of patients and to assist them in their understanding of the physiological mechanisms which govern peritoneal transport. These methods are based on the Pyle-Popovich peritoneal mass transport model which encompasses both diffuse and convective transport as well as lymphatic flow and residual renal function. Algebraic solutions to the mass balance equations governing solute transport are provided. Since these solutions are expressed explicitly as functions of time, they are easily programmed for use on a personal computer or calculator. This offers considerable advantage over the more computer-intensive numerical solutions which had been previously required since one can now calculate both mass removal and changes in blood concentration at the end of an exchange without requiring any intermediate calculations. This computational advantage and the ability to model changes in blood concentration are shown to be of particular importance when modeling more dynamic therapies such as CCPD or Tidal peritoneal dialysis. Finally, the model and solutions, when assessed clinically among 5 patients on two separate occasions, resulted in predicted fluid and mass removals which were in high concordance with measured fluid and mass removals (concordance correlation coefficients in excess of 0.97). Our findings suggest that kinetic modeling can provide the kind of analytical tools necessary to guide clinicians in their care and management of peritoneal dialysis patients.

This chapter will focus on techniques other than dialysis for removal of chemical toxins (endogenous or exogenous), immune toxins, or other naturally occurring biochemical substances considered to produce disease. Plasmapheresis is the process of removal of plasma (by mechanical, immunoprecipitation, cryoprecipitation, or filtration techniques) which contains the substance in question. Hemoperfusion is the passage of blood over sorbent agents for removal of harmful products

Not only have the number and type of hemodialyis membranes proliferated over the past decade, but expectations of what a membrane should, and should not, do have altered to the extent that researchers and clinicians alike now find it difficult to understand the patient consequences of hemodialyzer membrane selection. The associated literature is extensive, e.g. Medline® contains over 1500 citations to hemodialysis membranes, but several comprehensive technical reviews are available (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). This chapter begins with a brief introduction and history. Then it describes and classifies currently available membranes and summarizes our current understanding of how a membrane’s physical properties and chemical structure determine its transport characteristics and biocompatibility

This study explores the suitability of using encapsulated genetically modified fibroblasts for orthopedic tissue engineering by examining cell survival and persistence of human transforming growth factor-beta (hTGF-beta) overexpression in xenogeneic and allogeneic implant models. Human wild-type fibroblasts, modified to produce a latent form of hTGF-beta, and murine mutant-type fibroblasts, engineered to release a constitutively active form of hTGF-beta, were encapsulated separately in Ca2+ -alginate microcapsules. Following a percentage viability assessment by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test, microcapsules were implanted into either the subcutaneous or intraperitoneal cavities of mice. Explanted encapsulated cells were characterized for percentage viability and subjected to a release study and a viability test 1 week and 3 weeks following implantation, a time frame consistent with the requirement for orthopedic tissue engineering application of this growth factor. On average, percentage viabilities of encapsulated cells were 64%at implantation, 52% at explantation, and 56%after 1 week following either 1- or 3-week explantation. hTGF-beta release declined following in vivo implantation, more so for xenogeneic than allogeneic models, but remained in the clinically attractive range of 2 to 30 ng/(10(6) implanted cells 24 h). This technical platform for hTGF-beta is very encouraging for cartilage regeneration using orthopedic tissue engineering, and further evaluation is warranted.

Islet-like cell clusters (ILCCs) were derived from murine embryonic stem cells using a slightly modified version of the protocol originally described by Lumelsky et al. in 2001. Analysis with enzyme-linked immunosorbent assays (ELISAs) that distinguish human from murine insulin demonstrated that insulin released from these ILCCs, upon initial in vitro glucose challenge, was of non-murine origin and in fact corresponded to the species of insulin, human or bovine, that had been added to the culture media used to derive ILCCs. This finding convincingly supports the hypothesis that ILCCs are not synthesizing insulin de novo, but rather simply regurgitating insulin taken up during tissue culture. In further experiments, ILCCs were derived in media in which insulin had been replaced by IGF-I with which it shares a common signaling pathway. These ILCCs failed to release any detectable insulin. In contrast, ILCCs produced by various protocols stained positive (dithizone and immunoselective antibodies) for intracellular insulin and, in some cases, C-peptide. Despite the presence of at least some level of de novo, synthesized insulin in ILCCs, the majority of insulin released by ILCCs was sequestered from the exogenous medium.

This study was undertaken to develop an in situ source of transforming growth factor-beta1 (TGF-beta1), one of several molecules potentially useful for a tissue-engineered bioartificial cartilage. Primary human fibroblasts and murine NIH 3T3 cells were genetically modified via viral transfection to express human TGF-beta1. Two viral constructs were used, one expressing a gene encoding for the latent and the other for the constitutively active form of the growth factor. Unmodified cells served as controls. Four genetically modified cohorts and two controls were separately encapsulated in a 1.8% alginate solution using a vibrating nozzle and 0.15M calcium chloride crosslinking bath. Diameter of the spherical capsules was 410 +/- 87 microm. In vitro release rate measured over 168 hours varied with cell types and ranged from 2-17 pg/(milligram of capsules x 24 h) or 2-17 ng/(10(6) cells x 24 h). None of the formulations exhibited a large initial bolus release. Even when serum-supplemented medium was not replenished, cell viabilities remained over 55% after 1 week for all cell types. Microencapsulated genetically modified cells were capable of a constitutive synthesis and delivery of biologically significant quantity of TGF-beta1 for at least 168 hours and thus are of potential utility for artificial cartilage and other orthopedic tissue engineering applications.

This paper begins with an extensive review of previous research on the degradation of non-protein nitrogen compounds for improved therapy of renal failure. During the 1970s, Malchesky established that naturally occurring strains of microorganisms were highly effective for the in vitro degradation of urea and other compounds found in urine, and that these bacteria could be conditioned with selected media to enhance growth and degradation efficiency. A few years later, Setala introduced the concept of oral delivery of lyophilized bacteria, harvested from soil, to uremic patients, for degradation of non-protein nitrogen compounds. In the 1990s, Chang proposed delivery of encapsulated genetically modified bacteria for removal of uremic waste products in vitro and in vivo. Recently, our group has pursued the idea of orally delivering formulated combinations of enzymes or modified bacteria. A new study is also described, which characterizes the capacity of a single alginate microcapsule containing a mixture of genetically modified cells and enzyme to degrade urea, uric acid and creatinine. The combination capsules were found to be effective in vitro and in vivo in a rodent model of chemically-induced renal failure. Reduction of urea concentration in vivo required co-administration of a cation exchange resin to adsorb ammonia. Increased investigative effort is warranted for these approaches which offer significant potential as an adjunct to conventional forms of dialysis.

This study was undertaken to characterize the capacity of a combination of genetically modified bacteria to lower elevated levels of urea and uric acid and thus to serve as a potential adjunct to maintenance dialysis in patients with chronic renal failure. Two strains of genetically modified bacteria expressing enzymes, urease to degrade urea and uricase to degrade uric acid, were identified, combined, and dispersed in 600-microm alginate microcapsules suitable for oral administration. In 24 h in vitro experiments, 5 mL of these capsules completely cleared 95% of the urea and >99% of the uric acid from 100 mL of a challenge solution formulated to the concentration of these solutes in a presenting hemodialysis patient. The process of urea degradation was found to be intracellular and each bacterial strain was specific for its substrate. Solute degradation in vivo was evaluated with a chemically induced model of acute renal failure, using Sprague-Dawley rats. Orally administered capsules were found to remain in the gastrointestinal tract for at least 6 h. The severity of azotemia and hyperuricaemia after chemical induction of acute renal failure was reduced by 64 and 31%, respectively, on administration of the capsules. Reduction of urea concentration (but not uric acid concentration) in vivo required coadministration of an ion-exchange resin to adsorb ammonia. Oral delivery of a combination of genetically modified microorganisms should be further explored in chronic renal failure models as a useful adjunct to dialysis or to immunosorption for the treatment of uremia.

An alginate microcapsule was developed that contains three enzymes (urease, uricase, and creatininase) capable of effectively degrading urea, uric acid, and creatinine, which are elevated to pathologic levels in patients with kidney failure. The capsules were evaluated in vitro and in vivo in a rodent model and evidenced considerable potential as a possible adjunctive therapy in the treatment of ESRD. In vitro, 5 mL of the capsules incorporating a quantity of enzymes in the mg range effectively degraded all the uric acid, 97% of the urea, and 70% of the creatinine within 24 hours in a 100 mL test solution simulating the concentration of these solutes in uremic plasma. Enzyme degradation of urea followed Michaelis-Menten kinetics, and the Lineweaver-Burk plots for both encapsulated enzymes and unencapsulated control animals were superimposable, indicating that mass transfer through the capsules was not rate limiting in the degradation process. A chemically induced acute renal failure model in the rat was used to evaluate the ability of encapsulated enzymes, along with an oral sorbent (ion exchange resin), to degrade uremic toxins in vivo. Encapsulated enzyme therapy decreased the severity of azotemia by as much as 70%. Preliminary scale up calculations indicated that oral delivery to humans would involve a practical and manageable quantity of enzymes. This is the first study using a combination of enzymes in a single delivery vehicle to degrade multiple uremic toxins.

In the past 5 years, stem cells have emerged from condign obscurity to remarkable levels of prominence in both the scientific literature and lay consciousness. The potential therapeutic applications of stem cells span virtually every facet of regenerative medicine. Stem cell science and technologies are being pursued by the National Institutes of Health (NIH) and other government research agencies and in the private sector. This chapter focuses on the latter and is intended to quantify and discuss current commercial involvement in the development of stem cell technology. A list of qualifying firms is compiled from general awareness of the field, from keyword Web searches, from companies whose scientists presented at relevant technical or investor conferences and from the trade literature. The private sector is just one of many sources of stem cell research and development. Government agencies both perform work in their own laboratories and fund research in universities and research institutes. Foundations and non-for-profit laboratories also support stem cell research. No summary data on public sector spending on stem cells seems to be available. Based on the number and impact of publications in the peer-reviewed literature, it would appear that publicly supported stem cell research equals or exceeds that of the private sector.