Control of the timing and dosage of IGF-I delivery from encapsulated cells.

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 Ca²âº-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.

How to teach artificial organs.

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.

A localizable, biological-based system for the delivery of bioactive IGF-1 utilizing microencapsulated genetically modified human fibroblasts.

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.

Great expectations: private sector activity in tissue engineering, regenerative medicine, and stem cell therapeutics.

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.

Microencapsulated cells genetically modified to overexpress human transforming growth factor-beta1: viability and functionality in allogeneic and xenogeneic implant models.

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.

In vitro characterization of TGF-beta1 release from genetically modified fibroblasts in Ca(2+)-alginate microcapsules.

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.

Sequestration and synthesis: the source of insulin in cell clusters differentiated from murine embryonic stem cells.

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.

In vivo and in vitro degradation of urea and uric acid by encapsulated genetically modified microorganisms.

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.

Degradation of low molecular weight uremic solutes by oral delivery of encapsulated enzymes.

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.

Tissue engineering: the end of the beginning.

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.

Oral administration of biochemically active microcapsules to treat uremia: new insights into an old approach.

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.

Private sector development of stem cell technology and therapeutic cloning.

Based on data collected in June 2002, more than 30 biotechnology startup firms in 11 countries are pursuing commercial development of stem cell technology and therapeutic cloning. These firms employ 950-1000 scientists and support staff and spend just under $200 million on research and development each year. The field has the look and feel of a high-tech cottage industry, with about half the startups employing fewer than 15 FTEs (full time equivalents). Funding is mostly from venture capitalists and private investors. Participants are geographically dispersed, with about 40% of the activity outside the United States. Focus is equally split between embryonic and adult stem cells. Taken as a whole, both the structure and scope of the private sector in stem cell research seem appropriate to the promise and development time frames of this important new technology.

Kinetics and dosing predictions for daily haemofiltration.

BACKGROUND: Thrice-weekly haemofiltration affords excellent outcome when it is used to treat chronic renal failure patients. Daily haemofiltration (DHF) has recently been proposed as a more intensive therapy option, but the total ultrafiltration or exchange volume (replacement volume plus net ultrafiltration volume) requirements for adequate solute clearances during this novel therapy are unknown. METHODS: We calculated theoretical solute kinetic profiles during six times per week DHF for comparison with those during thrice-weekly haemodialysis using a high-flux dialyser (HFHD) or during continuous ambulatory peritoneal dialysis (CAPD). HFHD and CAPD were chosen for comparison because K/DOQI guidelines have defined adequate treatment doses for these therapies. Steady-state concentrations were calculated using a two-compartment model of an anuric patient with 35 l of total body water for five solutes: urea, creatinine, vitamin B(12), inulin and beta(2)-microglobulin. Solute distribution volumes and generation rates were taken from the literature, and excess fluid (1 l/day) was assumed to accumulate in and be removed from the extracellular fluid compartment. Theoretical predictions of solute clearance were compared for a 15-l exchange volume/session during DHF, urea Kt/V of 3.6/week during HFHD and urea Kt/V of 2.0/week during CAPD as solute-specific values of the equivalent renal clearance (EKR) and standard Kt/V (stdKt/V) recently defined by Gotch. Additional comparisons of solute clearances were performed between DHF and other daily therapies including six times per week short daily haemodialysis (SDHD) and six times per week nocturnal haemodialysis (NHD). RESULTS: The calculated results predict that: (i) urea clearance during DHF with an exchange volume of 90 l/week (6x15 l) is equivalent to those during HFHD and CAPD based on urea stdKt/V; and (ii) middle molecule clearances during DHF exceed those achieved during HFHD and CAPD based on either EKR or stdKt/V. As expected, DHF therapy was inferior regarding the clearance of urea and other small solutes to SDHD and NHD; however, DHF therapy was superior to SDHD regarding the clearance of larger middle molecules, approaching the clearances achieved by NHD. CONCLUSIONS: We predict that an exchange volume of approximately 40% of total body water (15/35 l=43%) per session will provide adequate clearance of small solutes and substantial clearance of middle molecules during six times per week DHF therapy. These theoretical predictions require clinical validation.

Maintenance dialysis population dynamics: current trends and long-term implications.

Despite a general recognition that treatment of end-stage renal disease (ESRD) has become a large-scale undertaking, the size of the treated population and the associated costs are not well quantified. This report combines data available from a variety of sources and places the current (midyear 2001) estimated global maintenance dialysis population at just over 1.1 million patients. The size of this population has been expanding at a rate of 7% per year. Total therapy cost per patient per year in the United States is approximately 66,000 dollars. Assuming that this figure is a reasonable global average, the annual worldwide cost of maintenance ESRD therapy in the year 2001, excluding renal transplantation, will be between 70 and 75 billion US dollars. If current trends in ESRD prevalence continue, as seems probable, the ESRD population will exceed 2 million patients by the year 2010. The care of this group represents a major societal commitment: the aggregate cost of treating ESRD during the coming decade will exceed 1 trillion dollars, a thought-provoking sum by any economic metric.