Tissue engineering: challenges and opportunities.

This article reviews the key developments in the tissue engineering field over the past several years. The issues related to the development of the components of tissue-engineered products including cells, biomaterials, and biomolecules, and their integration into safe and effective products are presented. Moreover, the article outlines the challenges to the commercialization of tissue-engineered products, and highlights the ongoing efforts by the American Society for Testing and Materials (ASTM) in developing standards for tissue-engineered medical products. Furthermore, funding opportunities at the Advanced Technology Program at NIST are presented. Published 2000 John Wiley & Sons, Inc.

Effects of particulate and soluble cadmium species on biochemical and functional parameters in cultured murine macrophages.

Cultured murine macrophages (RAW 264.7) were used to evaluate the temporal relationships between cytotoxicity, phagocytosis, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) production, and alterations in expression of stress proteins after exposure to cadmium oxide (CdO) or cadmium chloride (CdCl(2)), particulate and soluble forms of cadmium, respectively. Macrophages were exposed in vitro to CdO (25 or 50 microg) or CdCl(2) (30 or 40 microM) for 2 to 72 h. Cytotoxicity was not evident until 18 h when exposed to 30 microM CdCl(2) or 25 microg CdO, but occurred as early as 12 h after exposure to 40 microM CdCl(2) or 50 microg CdO. Relative to untreated controls, phagocytic activity decreased progressively from 2 to 24 h after exposure to both forms of cadmium. TNF-alpha levels increased to 2- to 3-fold after 4 h and remained elevated until 24 h after exposure to 25 and 50 microg CdO and 30 microM CdCl(2), but decreased by 18-24 h at 40 microM CdCl(2). CdCl(2) or CdO alone did not induce NO; however, both cadmium species reduced lipopolysaccharide (LPS)-stimulated NO production in a dose-dependent manner. Enhanced de novo synthesis of 70- and 90-kD heat shock, or stress, proteins was observed 2 to 8 h after exposure to both CdCl(2) and CdO; however, synthesis of these proteins returned to control levels by 24 h. Stress protein synthesis was enhanced by CdCl(2) or CdO prior to cytotoxicity, but coincided with a decrease in phagocytic capacity and an increase in TNF-a levels. The data suggest that cultured macrophages respond similarly in vitro to a particulate form and a soluble form of cadmium in a cell type that plays a pivotal role in inflammatory and immune responses.

Synergistic induction of tumor necrosis factor alpha by bacterial lipopolysaccharide and lipoteichoic acid in combination with polytetrafluoroethylene particles in a murine macrophage cell line RAW 264.7.

The induction of tumor necrosis factor alpha (TNF-alpha) by polytetrafluoroethylene (PTFE) particles (5-50 microns) and by bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) was examined in RAW cell cultures. Twenty-four-hour culture supernatants from the treated and control cells were assayed for TNF-alpha using a mouse L929 cell cytotoxicity assay. Untreated RAW cells produced low levels of endogenous TNF-alpha in the culture supernatants. Addition of 0.5 ng to 1 microgram/ mL LPS or 1 ng to 1 microgram/ml LTA increased the TNF-alpha production by 7-3570-fold and 2-815-fold, respectively. Addition of 1-5 mg PTFE increased the TNF-alpha production by 6-17-fold over the untreated control cell levels. The cells exposed to PTFE and 0.5 ng/mL LPS or 5 ng/mL LTA produced TNF-alpha levels that were significantly higher than those produced by any inducer alone. Thus, both LTA, a Gram-positive bacterial cell wall component and LPS, a Gram-negative bacterial cell wall component, can induce TNF-alpha production, which is further enhanced by PTFE particles in RAW cells.

Regulatory concerns in the development of biologic-biomaterial combinations. United States Food and Drug Administration.

Several biologic-biomaterial combinations are currently under development in an attempt to modulate tissue or organ function in patients. The FDA regulations on combination products and the intercenter agreements among the Center for Biologics Evaluation and Research (CBER), the Center for Devices and Radiological Health (CDRH), and the Center for Drugs Evaluation and Research (CDER) provide further guidance on center jurisdiction of combination products and other products where there are jurisdictional concerns. The biological component of biologic-biomaterial combinations raises a number of issues that relate to the safety and bioactivity of the final product. For example, transmission of adventitious agents to patients via somatic cells, tissue, or cell-derived products is a major safety concern as are in vivo inflammatory responses elicited by the biomaterial component. CBER has drafted a number of "Points to Consider" documents to provide further guidance in the development of biological products. The intent of this article is to provide the highlights of the FDA regulations for combination products and the intercenter agreement between CBER and CDRH delineating the responsibilities of each center for medical device activities. In addition, the article focuses on the CBER's concerns related to the development of somatic cell-biomaterial combinations for therapeutic use.

Regulatory concerns in the development of topical recombinant ophthalmic and cutaneous wound healing biologics.

The use of recombinant DNA technology has enabled the development of an increasing number of endogenous growth regulatory peptides for potential use as therapeutic biologics. Numerous such recombinant peptides are now licensed, and many are in various stages of pharmaceutical development. Although currently there are a number of "Points to Consider" and related guidance documents available concerning various issues of biotechnology-derived products, the purpose of this article is to focus on the use of these biologics in topical ophthalmic and chronic cutaneous wound healing. Regulatory expectations with respect to product quality, safety, and efficacy that may be particularly germane to these products will be discussed. Providing regulatory guidance on these issues may not only facilitate the introduction of safe and effective new biologic therapies into clinical trials at the investigational level but also provide appropriate information to aid in their eventual approval for licensure and widespread clinical use.

FDA regulations for growth factors and related products.

Biological products, including the majority of growth factors, are regulated by the Center for Biologics Evaluation and Research (CBER) in the U.S. Food and Drug Administration (U.S.FDA) under two statutes; U.S. Federal Food Drug and Cosmetics (FDC) act and the U.S. Public Health Service act. As stipulated in the U.S. Code of Federal Regulations (21 CFR) under the FDC act, the testing of new products in humans is conducted under an Investigational New Drug (IND) application. The primary objective of the FDA in all Phases of an investigation is assure safety of human subjects. During phase II and phase III studies, additional assurance regarding the scientific quality of the clinical investigation is required. A marketing approval is granted by CBER following the review of Product License Applications (PLA) and Establishment License Applications (ELA). CBER's review process provides guidance to the manufacturers of biological products towards the development of safe and effective biological products for human use. Information pertinent to preclinical issues and clinical trial design is presented here with a special emphasis on the non-hematopoietic growth factors.

Dexamethasone prevents the growth inhibitory effects of recombinant tumor necrosis factor in a rat hepatoma cell line Reuber-RC-3: an association with the changes in the messenger RNA levels for multidrug resistance gene.

Two days exposure to recombinant tumor necrosis factor (rTNF-alpha) produced a dose-dependent reduction in (methyl-3H) thymidine incorporation in RC-3 cells (ID50 = 25 units/ml). Prolonged treatment with rTNF-alpha further resulted in a significant reduction in colony formation (ID50 = 200 units/ml), which was reversed upon removal of the agent. Interferon levels were undetectable in the supernatants of the rTNF-alpha treated cells. Simultaneous exposure to dexamethasone prevented the growth inhibition in rTNF-alpha-treated RC-3 cells. Significant dose-dependent increase in the steady state levels of the mRNA for multidrug resistance (MDR1) gene was observed after rTNF-alpha treatment while simultaneous exposure to dexamethasone produced a substantial reduction in the mRNA levels for MDR1 gene. These data suggest that growth inhibitory effects of TNF are regulated by dexamethasone and are associated with changes in MDR1 mRNA levels in hepatoma-derived cells.

Isolation and characterization of a rat liver epithelial cell line resistant to the antiproliferative effects of transforming growth factor beta (type 1).

Rat liver epithelial cells resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF-beta 1) were isolated after 3 h exposure to 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine followed by continuous treatment with 1 ng/ml TGF-beta 1 for 6 weeks. In comparison to the parental or N-methyl-N'-nitro-N-nitrosoguanidine-exposed rat liver epithelial cells (concentration causing 50% inhibition of the rate of DNA synthesis, 0.25 ng/ml), these cells were 10-fold more resistant to the antiproliferative effects of TGF-beta 1 and exhibited resistance to growth inhibition by a highly purified liver-derived growth inhibitor, recombinant human tumor necrosis factor, and transforming growth factor beta 2. Single cell cloning of these resistant cells led to the isolation of a nontransformed clonal cell population (clone 11) which maintained stable resistance in the absence of TGF-beta 1 treatment. Binding of 125I-labeled TGF-beta 1 to rat liver epithelial cells and clone 11 cells was similar. Clone 11 cells exhibited a 5-10-fold resistance to the cytotoxins Adriamycin and vinblastine as assessed by a clonogenic assay. This drug resistance was accompanied by an increase in the steady state levels of the mRNAs for multidrug resistance gene (MDR-1), glutathione S-transferase-P, TGF-beta 1, and c-myc genes. The data presented here suggest an association between resistance to the growth-inhibitory effects of TGF-beta 1- and MDR-1-mediated multidrug resistance.

Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor beta 1, and recombinant tumor necrosis factor alpha, in normal and neoplastic cells.

The growth modulatory effects of a rat liver-derived growth inhibitor (LDGI), transforming growth factor beta 1 (TGF-beta 1), and recombinant tumor necrosis factor (rTNF-alpha) were examined in a variety of liver-derived and nonliver-derived normal and neoplastic cell culture systems. Normal rat liver epithelial (RLE) cells were highly sensitive to the growth inhibitory effects of LDGI (ID50 = 0.2 ng/ml) and TGF-beta 1 (ID50 = 0.25 ng/ml) but were less sensitive to rTNF-alpha (ID40 = 5000 Units/ml). Aflatoxin B1-transformed RLE cells showed sensitivity to the cytostatic effects of LDGI (ID50 = 1.5 ng/ml); however, these cells were completely resistant to the antiproliferative effects of TGF-beta 1 and rTNF-alpha. Clones isolated from these transformed cells, exhibited a wide range of sensitivities to LDGI but all of the clones were resistant to the growth inhibitory effects of both TGF-beta 1 and rTNF-alpha. Rat hepatoma Reuber cells were extremely sensitive to the antiproliferative effects of rTNF-alpha (ID50 = 10 Units/ml) but exhibited sensitivity to LDGI only at concentrations above 1.5 ng/ml and were resistant to the antiproliferative effects of TGF-beta 1. Rat hepatoma UVM 7777 cells and human hepatoma HepG2 cells, however, were insensitive to the growth inhibitory effects of all three factors. Among the nonliver-derived cells, human breast carcinoma (MCF-7) cells were extremely sensitive to rTNF-alpha (ID50 = 20 Units/ml, exhibited some sensitivity to LDGI (ID50 = 1 ng/ml), and were resistant to the antiproliferative effects of TGF-beta 1. In contrast, the rate of DNA synthesis is rat kidney fibroblasts and human foreskin fibroblasts was significantly stimulated in response to TGF-beta 1, LDGI, and rTNF-alpha. These data demonstrate that LDGI, TGF-beta 1, and rTNF-alpha exert positive and negative modulations of growth in different cell systems and that the growth regulatory effects of LDGI differ from those of TGF-beta 1 and rTNF-alpha in some cell types.

The epidermal growth factor- and interferon-independent effects of double-stranded RNA in A431 cells.

The human epidermoid carcinoma cell line A431, containing an amplification in the epidermal growth factor (EGF) receptor gene, was examined for its sensitivity to the growth inhibitory effects of synthetic double-stranded RNAs (dsRNAs). Poly(I).poly(C), poly(A).poly(U) and rln.r(C13,U)n at 5 to 100 micrograms/ml produced 20 to 60% growth inhibition, whereas poly(ICLC) produced 40 to 80% growth inhibition at 0.05 to 25 micrograms/ml.Poly(I).poly(C) did not cause the secretion of interferon (IFN) into the medium, and addition of polyclonal antibodies to IFN-alpha and IFN-beta did not block the growth inhibition produced by poly(I).poly(C). Clone 29, which proliferates in response to EGF, and clone 29R, which is sensitive to the growth inhibitory effects of EGF, showed sensitivities to the antiproliferative effects of poly(I).poly(C) similar to those of the parent cell line. Incubation of cell membrane extracts with poly(I).poly(C) or treatment of cells with the dsRNA did not affect EGF receptor tyrosine kinase activity. On the other hand, poly(I).poly(C) produced a dose-dependent induction of (2',5')oligo(A) synthetase activity and degradation of 45S preribosomal RNA and 28S and 18S rRNA. These results indicate that the growth inhibitory properties of poly(I).poly(C) in A431 cells are independent of the action of IFN but are associated with degradation of rRNA, an effect that may be related to the (2',5')oligo(A)-RNase L pathway.

The synergistic cytocidal effect produced by immune interferon and tumor necrosis factor in HT-29 cells is associated with inhibition of rRNA processing and (2',5') oligo (A) activation of RNase L.

The cytocidal activity of human immune interferon (IFN-tau) in combination with recombinant tumor necrosis factor-alpha (TNF) was assessed in human colon carcinoma cell line HT-29. IFN-tau or TNF alone reduced cell viability by 15-20%, but in combination produced a synergistic cytotoxic effect which inhibited colony formation by 55-85%. TNF impaired the processing of ribosomal precursor 45S RNA to 32S RNA which resulted in the selective inhibition of 28S rRNA. Both TNF and IFN-tau induced the activity of (2',5') oligo (A) synthetase and produced selective degradation of 28S rRNA, effects possibly related to the (2',5') oligo (A)-dependent activation of RNase L. The combination of IFN-tau and TNF resulted in a marked reduction in the labeling of ribosomal precursor RNAs as well as mature rRNA. These results demonstrate that the posttranscriptional and processing effects of IFN-tau and TNF are related to the enhanced cytotoxicity by this combination of cytokines.

Synergistic effect of retinoic acid and calcium ionophore A23187 on differentiation, c-myc expression, and membrane tyrosine kinase activity in human promyelocytic leukemia cell line HL-60.

The effect of the combination of retinoic acid (RA) and calcium ionophore A23187 on cellular differentiation was assessed in promyelocytic leukemia cell line HL-60. RA (10(-10)-2.5 X 10(-8) M) or A23187 (4 X 10(-7) M) alone produced 15-22% differentiated cells as assessed by nitroblue tetrazolium reduction. Exposure of cells for 48 hr to the combination of 4 X 10(-7) M A23187 and 10(-10)-2.5 X 10(-8) M RA resulted in 20-86% of the cells capable of reducing nitroblue tetrazolium, but with no measurable level of nonspecific esterase activity. The combination of A23187 and either dimethyl sulfoxide, 1,25-dihydroxyvitamin D3, or immune interferon failed to produce a synergistic effect on differentiation. Addition of either the calmodulin antagonists, N-(6-aminohexyl)-5-chloronaphthalenesulfonamide and trifluoperazine, or the protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, during treatment with A23187 and RA did not block differentiation. Membrane tyrosine kinase activity was measured in cells treated with A23187 and RA in a nondenaturing gel system using the exogenous substrate poly(Glu:Tyr). Membrane-bound tyrosine kinase activity was not present in untreated or RA-treated cells but was induced by A23187 treatment alone and was markedly increased in cells 48 hr after treatment with the combination of A23187 and RA. Significantly greater reduction in c-myc mRNA levels was also observed 24 hr after treatment with RA and A23187 in comparison to that observed with either agent alone. These results suggest that a Ca2+-mediated process sensitizes cells to the differentiating effect of RA and that this effect is associated with a significant reduction of c-myc expression and the induction of membrane tyrosine kinase activity in this cell line.

Appearance of membrane-bound tyrosine kinase during differentiation of HL-60 leukemia cells by immune interferon and tumor necrosis factor.

The effect of immune interferon (IFN-gamma) and recombinant tumor necrosis factor (rTNF-alpha) on cellular differentiation was investigated in human promyelocytic leukemia cell line HL-60. Both IFN-gamma and rTNF-alpha induced the appearance of the monocytic phenotype in a dose- and time-dependent manner as assessed by morphology, reduction of nitroblue tetrazolium and the induction of alpha-naphthyl butyrate esterase. Utilizing a nondenaturing polyacrylamide electrophoretic assay, it was revealed that a membrane-bound tyrosine kinase activity accompanied the appearance of the differentiated cell type. These results suggest that the induction of membrane-bound tyrosine kinase activity by IFN-gamma and rTNF-alpha may be an important characteristic of monocytic differentiation.

Potentiation of the cytocidal effect of human immune interferon by different synthetic double-stranded RNAs in the refractory human colon carcinoma cell line BE.

A human cell line BE, derived from an undifferentiated carcinoma of the colon, was studied for its response to the cytocidal effects of human immune interferon (IFN-gamma) alone and in combination with various double-standard RNAs (dsRNAs). BE cells were moderately refractory to 3-day treatment with IFN-gamma (10 to 300 units/ml) where only 5 to 30% reduction in colony formation occurred. A similar exposure interval to polyriboinosinic.polyribocytidylic acid [poly(I).poly(C)] (100 micrograms/ml) had no detectable effect on colony formation. In contrast, the lethal effect of the combination of IFN-gamma and poly(I).poly(C) was synergistic and this regimen produced a 40 to 80% reduction in colony formation. The cytocidal effects of the combination of IFN-gamma with varying concentrations of the dsRNAs poly(I).poly(C), polyriboadenylic.polyribouridylic acid [poly(A).poly(U)], polyriboinosinic.polyribocytidylic acid stabilized with poly-L-lysine in carboxymethylcellulose [poly(ICLC)], and mismatched dsRNA [rIn.r(C13,U)n] were also examined. The concentration of the dsRNAs producing a 50% decrease in cell viability in combination with IFN-gamma (100 units/ml) was 6 micrograms/ml for poly(I).poly(C), 1 microgram/ml for poly(A).poly(U), 3 ng/ml for poly(ICLC), and 16 micrograms/ml for rIn.r(C13,U)n. DNA, RNA, and protein synthesis in IFN-gamma and poly(I).poly(C)-treated cells were reduced in a dose-dependent manner. However, there were no changes in either (2',5')oligoadenylate concentrations or in ribosomal RNA transcription following treatment with IFN-gamma and poly(I).poly(C). Thus, the synergism resulting from the combination of IFN-gamma and dsRNA appears to be mediated via another, as yet unknown, mechanism.

Synergistic antitumor effect of fluoropyrimidines and polyinosinic-polycytidylic acid against L1210 leukemia.

The effect of polyriboinosinic X polyribocytidylic acid [poly(I) X poly(C)] on the antitumor activity of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) was evaluated in mice bearing L1210 leukemia. Coadministration intravenously of poly(I) X poly(C) and either FUra or FUrd on days 1,5, and 9 to mice bearing L1210 leukemia implanted subcutaneously resulted in a 40% greater increase in life span at the optimal antitumor dose versus FUra and FUrd alone. This effect appeared to result from greater host tolerance of a dose of FUra or FUrd which would otherwise be cytotoxic. The protective effect of poly(I) X poly(C) was also evident in non-tumor-bearing mice, as well as following administration of drug intraperitoneally to mice bearing the tumor implanted intraperitoneally. FUrd incorporation into RNA in the spleen, bone marrow, and small intestine revealed little or no changes after coadministration of poly(I) X poly(C). (2', 5')Oligo(A) synthetase activity, an indication of interferon activity, was markedly depressed in the spleen and bone marrow following treatment with FUrd; however, poly(I) X poly(C) administered together with FUrd returned (2', 5')oligo(A) synthetase activity to normal levels. These data indicate that poly(I) X poly(C) ameliorates the host toxicity of fluoropyrimidines, possibly via an interferon-mediated effect, and thereby results in enhanced therapeutic efficacy of the antimetabolite as an antitumor agent.

Synergistic effect of human immune interferon and double-stranded RNA against human colon carcinoma cells in vitro.

The cytocidal activity of human immune interferon (IFN-gamma) in combination with the synthetic double-stranded RNA, poly(I).poly(C), was investigated in human colon carcinoma cell line HT-29. Three days of treatment with IFN-gamma (10 to 25 units/ml) resulted in 30 to 40% reduction in colony formation, whereas poly(I).poly(C) (25 to 100 micrograms/ml) reduced cell viability by 10 to 20% of control. The lethal effect of the combination of IFN-gamma and poly(I).poly(C) was synergistic wherein 70 to 90% reduction in colony formation was observed. Measurements of DNA, RNA, and protein synthesis after IFN-gamma and poly(I).poly(C) treatment showed a dose-dependent reduction in all three parameters. Recombinant IFN-gamma in combination with poly(I).poly(C) exhibited a similar effect. Studies evaluating the molecular mechanism of IFN-gamma and poly(I).poly(C) toxicity indicate a lack of involvement of the double-stranded RNA-dependent (2',5')oligoadenylate-RNase L and protein kinase pathways; however, the effect appears to be related to the inhibition of ribosomal RNA transcription in this cell line.

Evidence that high mobility group protein 17 is not phosphorylated in human colon carcinoma cells.

The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [32P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with 32Pi, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [gamma-32P]ATP.

Effects of human immune interferon on cell viability, (2',5')oligoadenylate synthesis, and polyamine-dependent protein phosphorylation in human colon carcinoma cells in vitro.

The growth-inhibitory effect of human immune interferon (IFN-gamma) was investigated in human colon carcinoma cell line HT-29. Three-day treatment of HT-29 cells with IFN-gamma (10 to 200 units/ml) resulted in 30 to 90% growth inhibition and 40 to 99% reduction in colony formation. Measurement of DNA, RNA, and protein synthesis following IFN-gamma treatment showed a dose-dependent reduction in all 3 parameters. The associated changes in (2',5')oligoadenylate [(2',5')oligo(A)] pathway were measured under growth-inhibitory conditions. Upon 1-day exposure to 25 to 200 units/ml of IFN-gamma, (2',5')oligo(A) synthetase activity was induced 10- to 15-fold and remained elevated for 3 days, whereas (2',5')oligo(A) phosphodiesterase activity remained unchanged. There was no detectable increase in intracellular (2',5')oligo(A) levels after IFN-gamma treatment, and ribosomal RNA degradation was not observed. Accompanying 1-day treatment with IFN-gamma (100 units/ml) was an induction of a polyamine-dependent protein kinase, which was double-stranded RNA-independent and phosphorylated endogenous polypeptides with molecular weights of 68,000 and 72,000. A similar exposure of cells to IFN-gamma (25 to 100 units/ml) resulted in 30 to 70% inhibition of ornithine decarboxylase activity; however, no significant alteration in intracellular polyamine levels was observed. These data suggest that IFN-gamma-dependent toxicity is not related to (2',5')oligo(A) activation of a latent endoribonuclease but is accompanied by protein phosphorylation, which is, in part, stimulated by exogenous polyamines.