Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Atualmente, grande parte das investigaÃÃes sÃo dirigidas para a anÃlise de mediadores da resposta imuno-inflamatÃria local e sistÃmica apÃs a agressÃo tÃrmica. A Mitomicina-C (MMC), um antibiÃtico isolado do Streptomyces caespitosus, à utilizado clinicamente para diminuir o processo de cicatrizaÃÃo de feridas, devido a sua aÃÃo antiproliferativa em fibroblastos in vitro. Produziram-se queimaduras dÃrmicas em quarenta e oito camundongos Swiss albino utilizando-se um ferro de solda modificado, aplicado à pele por nove segundos. No primeiro dia (DO) a queimadura foi submetida a tratamento tÃpico, em dose Ãnica, com SF 0,9% (controle) ou Mitomicina-C (MMC). Avaliaram-se os efeitos da MMC na cicatrizaÃÃo de queimaduras utilizando-se mÃtodos macroscÃpicos, microscÃpicos e computacionais no 4 dia (D4), 7o (D7), 14o (D14) e 21o pÃs-queimadura (D21PQ). Na anÃlise macroscÃpica, utilizou-se escala visual analÃgica (EVA), e planimetria digital; nas microscÃpicas, as amostras de pele foram coradas pela picrosirius red (PR) analisadas à microscopia de luz polarizada, quantificando-se a densidade do colÃgeno tipo I e tipo III. Os parÃmetros biomÃtricos nÃo evidenciaram efeitos deletÃrios sobre o estado nutricional dos animais. A EVA demonstrou que as feridas do grupo MMC exibiram melhor aparÃncia significativa que as do grupo controle no D14PQ com p= 0,0002. A taxa mÃdia de migraÃÃo das margens das feridas (TMM) evidenciou menor valor no grupo MMC, sendo diferenÃa bastante significativa (p =0, 0033). A morfometria do processamento de imagem assistida por computador evidenciou que o colÃgeno tipo I teve um comportamento decrescente de deposiÃÃo no grupo controle; jà no grupo tratado com MMC, foi crescente entre D4 e D14, decrescendo entre D14 e D21. Da mesma forma constatou-se uma diferenÃa significante entre os grupos no D14 e bastante significante no D21 na quantidade de colÃgeno tipo III das feridas do grupo MMC, o que evidencia a capacidade da MMC de retardar a transformaÃÃo do colÃgeno imaturo (tipo III) em maturo (tipo I). A MMC foi eficaz em retardar a maturaÃÃo da ferida por queimadura tÃrmica, gerando menor quantidade de fibrose. / Many research works which are currently being carried out are driven towards the analysis of the mediators of the local and systematical immune inflammatory response after episodes of thermal aggression. Mitomycin-C (MMC), an insulated antibiotic of the Streptomyces caespitosus kind is used clinically in order to shorten the wound healing process due to its anti-proliferation action in fibroblasts in vitro. Dermal burns were produced on forty eight Swiss albine mice as a result of using an adapted iron-welding device which inflicted burns upon the back dorsum of the cavies for a lapse of time of nine seconds. On the first day the burns received topical treatment in a single dose with 0.9% sterile isotonic saline (control) or Mitomycin-C (MMC). The effects of MMC in the healing process of the burns were evaluated by using macroscopic, microscopic and computerized methods on the 4th day (PBD 4), 7th (PBD 7), 14th (PBD 14) and on the 21st post-burn day (PBD 21). For the macroscopic analysis an analogical visual scale (AVS) and a digital planimetry were used. On the microscopic analysis, the samples of the skin were colored by picrosirius red (PR) and analyzed at a microscope under a polarized light and the quantification of the collagen type I and III was carried out. The biometric parameters did not evidence any harmful effects over the nutritional conditions of the animals. The AVS evidenced that the wounds of the MMC group presented a significant better look than those of the control group in the PBD 14 (p= 0.0002). The wound edge migration rates (WEMR) evidenced a smaller rate in the MMC group than those of the control group, with significant difference (p =0.0033). The morphometry of the image processing, computer-assisted showed that the collagen type I presented a decreasing behavior in the process of healing in the control group, whereas in the group which was treated with MMC there was growth between PBD 4 and PBD 14 and a decrease between PBD 14 and PBD 21.Likewise, there was a significant difference among the groups on PBD 14 and a quite significant difference on PBD 21 in the amount of collagen type III on the wounds of the MMC, which evidences the capacity of MMC in slowing down the transformation of the immature collagen (type III) into a mature one (type I). MMC has proved to be efficacious in slowing the maturation of the wounds caused by thermal burns, thus generating a minor amount of fibrosis.

Heparan sulphate proteoglycans (HSPGs) are cell surface/secreted molecules expressed by all cells. HSPGs consist of carbohydrate side-chains attached to a core protein and are involved in regulating key signalling pathways in the developing mammalian brain via sugar-protein interactions. It has been hypothesized, in the ‘heparan sulphate (HS) code hypothesis’, that the specificity for the interaction between the HSPGs and particular signalling pathways is encoded by its HS side-chain. HS has an enormous variety of structures due to postsynthetic modification. Hs2st and Hs6st1 are enzymes involved in generating different HS structures by sulphating the 2-carbon or 6-carbon molecule of the sugar backbone respectively. Fibroblast growth factors (Fgfs) are a family of signalling molecules crucial for forebrain development. Some of its members such as Fgf8 are morphogens which pattern the forebrain via regulated gradient formation while others such as Fgf2 drive neurogenesis and cell proliferation. One of the main molecular consequences of Fgf signalling is activation of extracellular signal-regulated kinase (Erk) where the activation of Erk then drives developmental events such as neurogenesis or cell migration. Based on previous studies on the HS code hypothesis, we hypothesized that differential sulphation regulates Fgf signalling in a specific manner depending on the HS sulphation pattern. We performed binding assays on Hs2st-/- mice to ascertain the molecular mechanism behind the role of differential sulphation in Erk signalling through Fgf2 in the forebrain. We found that differential sulphation also has an important role to play in regionally targeting Fgf2/Erk signalling through regulating the formation of active signalling complexes. Studying the Fgf8/Erk signalling axis at E14.5 developing mouse corticoseptal boundary (CSB) revealed increased Fgf8 levels and Erk hyperactivation in both Hs2st and Hs6st1 null mutants. The dysregulation of Fgf8/Erk signalling at the CSB also highly correlates with the high expression of Hs2st and Hs6st1 at the CSB. A closer look into the molecular phenotypes of Hs2st-/- and Hs6st1-/- CSB revealed differences between them in which Hs6st1-/- CSB has higher Fgf8 levels compared to Hs2st-/- CSB. To elucidate the mechanisms underlying Hs2st and Hs6st1 role at the CSB, we investigated the formation and interpretation of Fgf8/Erk signalling gradient using Fgf8 bead assays in mice with Hs2st and Hs6st1 loss of function throughout development. We found that differential sulphation has a complex effect on Fgf8 gradient formation and interpretation in the forebrain in which Hs2st acts to stabilise the Fgf8 distribution through regulating Fgf8 levels through time while Hs6st1 acts to stabilise the Fgf8 distribution by maintaining the shape of the Fgf8 gradient through restricting Fgf8 levels during the formation of the Fgf8 distribution. In addition, we found Hs2st and Hs6st1 both function to increase the sensitivity of the CSB to Fgf8 for an Erk response although through different modes of action. Therefore, we conclude that differential HS sulphation plays a specific role in Fgf/Erk signalling depending on the HS sulphation pattern.

A organização de fibras de colágeno no leito de uma ferida é componente importante da cicatrização e contração da ferida, determinando em última instância a qualidade final da cicatriz. Neste estudo realizamos a implantação de modelo de biotecnologia constituído de géis de colágeno povoados por fibroblastos humanos, que foi utilizado como instrumento para a melhor compreensão dos fenômenos ainda pouco elucidados, envolvidos na contração de feridas. Utilizando fibroblastos procedentes de pele normal ou quelóides, observou-se maior contração dos géis povoados por fibroblastos oriundos de quelóide. O modelo implementado foi considerado eficiente para a avaliação da presença de moduladores da fase de remodelação da cicatriz, tais como o Fator de Crescimento Transformador Beta (TGF beta). A comparação entre a curva de contração gerada por fibroblastos oriudos de pele normal sob o efeito do TGF beta e a contração gerada por fibroblastos de quelóides, demonstra que as mesmas apresentam comportamento igual do ponto de vista estatístico. O modelo proposto demonstrou ser adequado para a melhor compreensão dos mecanismos responsáveis pela contração de feridas, bem como possui potencial na avaliação de novas drogas capazes de modular este fenômeno / An important component of tissue healing and wound contraction is the re-arrangement of ground collagen fibers, which can ultimately influence the final quality of scars. In this study we used a biotechnology experimental model with contracting collagen gels seeded with human fibroblasts in order to better understand the phenomena involved in wound contraction. We compared the contraction of the collagen gels using fibroblasts from normal skin and from keloids, and we observed that the collagen gels seeded with keloid fibroblasts suffered a bigger contraction. The model was considered efficient to test growth factors with the potential to modulate the remodeling phase of the scar, for example, the Transforming Growth Factor Beta (TGF beta). The analysis of the changing macroscopic gel area comparing the contraction generated by the normal fibroblasts after the treatment with TGF beta with the contraction in the gels with keloid\'s fibroblasts showed that they have the same behavior. The experimental model proved to be an useful tool to better understand the wound contraction and to test new drugs to modulate this phenomenon.

Experimentelle FACS-Flow-Analysen im Kontext einer radiogenen Lungenfibrose zur Veränderung der Zellproliferation von Maus-Lungen-Fibroblasten unter dem Einfluss von Kulturüberständen bestrahlter Fibroblasten. Additiv einzelne Versuche mit Antikörperzugabe gegen TGF-beta zur Evaluation eines hemmenden Effektes auf eine postulierte Arretierung der Fibroblasten in der G1-Phase der Zellteilung durch die Zytokine der Kulturüberstände bestrahlter Maus-Lungen-Fibroblasten. / FACS-Flow-Analyses on changes of cell-proliferations of mouse-lung-fibroblasts under the influence of culture-supernatants of irradiated fibroblasts in the context of a radiogenic lung fibrosis. Additionally some studies on a postulated inhibitory effect of an tgf-beta-antibody on the g1-phase cell-cycle-arrest of mitosis through cytokines in the supernatants of the irradiated fibroblasts.

Der wesentliche Dosis limitierende Faktor einer Strahlentherapie thorakaler Malignome ist die Strahlenempfindlichkeit des Lungenparenchymes, da sich mit einer Häufigkeit von 25-75 % aller Patienten ein Strahlenschaden des Lungengewebes entwickeln kann. Die Inzidenz einer Lungenfibrose nach 6- 12 Monaten liegt bei 15-30%. Die Kombination zytostatischer Medikamente mit ionisierender Strahlung kann die Ansprechraten verbessern, kann andererseits die Inzidenz einer Pneumonitis erhöhen. Die konkreten Mechanismen, die zu einer Pneumonitis und einer strahleninduzierten Fibrose führen, sind bislang noch nicht vollständig bekannt. Es wird vermutet, daß die ortsständigen Zellen der Lunge eine aktivere Rolle in der Pathogenese als bisher angenommen, einnehmen. Tiermodelle der Strahlenschädiung der Lunge zeigten ein sehr frühe Expression von TGF-ß-mRNA and fibronectin-mRNA nach Bestrahlung. TGF-ß und Fibronectin sind in der BALF und Serum von an thorakalen Malignomen erkrankten, strahlentherapeutisch behandelten Patienten erhöht. Neben Makrophagen und Typ II Pneumocyten als zelluläre Quellen der genannten Cytokine, sind Fibroblasten in der Lage beide Agentien in erheblichem Umfang zu synthetisieren. Ziele Um die aktive Rolle von Fibroblasten in der Pathogenese der strahleninduzierten Lungenfibrose in Abwesenheit von Entzündungszellen zu untersuchen, bestrahlten wir Lungenfibroblasten in vitro und beobachteten folgende Parameter. 1. Zellwachstum 2. Synthese von Fibronectin 3. Synthese von Kollagen ( Procollagen-I-Peptid) 4. Synthese von TGF-ß1 Methoden Humane fetale Lungenfibroblasten (MRC-5 ,ICN Biochemicals Eschwege ,Deutschland) wurden in DME Medium kultiviert unter Zugabe von 10% FCS plus L-Glutamine, Penicillin G , Amphotericin B und Gentamycin; Luftfeuchtigkeit 100% , Temperatur 37°, CO2 5%, Medienwechsel erfolgten zweimal wöchentlich und 24 Stunden vor den Messungen. 24h nach der Aussaat der Zellen erfolgte die Strahlenapplikation (CO 60; 4.5, 7.5, 10.5 Gy ). Messungen erfolgten an den Tagen 3,6,9,12,15 nach Bestrahlung. Hierfür wurden folgende Materialien verwandt. Fibronectin (ELISA), Takara TGF beta (ELISA), DPC Biermann Procollagen-I-Peptide (ELISA), Takara LDH ( kinetischer Assay), Sigma Cell counts (Zählkammer) Alle Messungen wurden zweimal unternommen. Ergebnisse: 1. Das Zellwachstum wurde dosisabhängig gehemmt. 2. Beginnend am 3 Tag stieg die Syntheserate des Fibronectin dosisabhängig. 3. Ähnliche Beobachtungen wurde bzgl der Procollagen-I-Peptid Synthese beobachtet. 4. TGF-ß Spiegel fanden sich nach Bestrahlung ab Tag 6 bis zum 4-fachen über dem Ausgangswert erhöht und kehrten ziwschen den Tagen 9 und 15 auf das Ausgangsniveau zurück. 5. Eine Erhöhung des LDH wurde nicht beobachtet. Dies zeigte, dass eine Zytolyse kein wesentlichen Einfluß hatte. Disskusion: Bei Bestrahlung humaner fetaler Lungenfibroblasten wird das Zellwachstum dosisabhängig limitiert. Dies wurde nicht durch einen strahlenbedingt erhöhten Zelltod hervorgerufen , da das bestimmte LDH ( ein Marker der Zytolyse) in den Zellkulturüberständen nicht erhöht war. Wir vermuten, das durch Bestrahlung eine Differenzierung von Progenitor Fibroblasten zu postmitotischen Fibrocyten erfolgte, wie auch bereits von anderen Arbeitsgruppen berichtet. TGF-ß fand sich nach Bestrahlung in den Zellkulturüberständen deutlich erhöht. Es wird angenommen , daß TGF-ß eine Schlüsselrolle in der Pathogenese fibrosierender Erkrankungen der Lunge, der Leber, der Niere spielt und ebenso in die Enstehung der durch ionisierende Bestrahlung hervorgerufene Lungenfibrose eingebunden ist. Unsere Experimente haben gezeigt , daß Fibroblasten in der Lage sind große Mengen TGF-ß and Fibronectin - sogar in Abwesenheit von Entzündungszellen- zu erzeugen und sich vermutlich autokrin stimulieren können. Dieser Mechanismus wird als wichtiger Co-Faktor in der Pathobiologie verschiedener zur Fibrose führender Lungenerkrankungen angenommen. Schlussfolgerung Fibroblasten produzieren erhöhte Mengen TGF-ß und Fibronectin nach Applikation ionisierender Strahlung. Sie könnten in der Pathogenese der Strahlenschädigung der Lunge eine aktivere Rolle spielen als bisher angenommen. / Introduction The major dosis limiting factor of radiation therapy of thoracic malignomas is the lung which may develop radiation injury with a frequency of 25-70% of patients .The incidence of lung fibrosis after 6-12 months ist 15-30 %. Combination of cytostatic drugs with ionizid radiation can improve response rates, but may result in a higher incidence of pneumonitis. The exact mechanisms leading to pneumonitis and radiation induced fibrosis of the lung are yet unknown.The structural cells of the lung are of the lung are probably involved in the pathogenesis in a more active way than thougt until now. Animal models of radiation injury of the lung showed a very early expression of TGF-beta -mRNA and fibronectin-mRNA after irradiation. TGF-ß and Fibronectin were elevated in BALF and in serum. Macrophages and type-II-pneumocytes are thought to be the cellular source, but fibroblasts also are capable to synthesize both agents in large amounts. Aims In order to investigate the active role of fibroblasts in the pathogenesis of radiation fibrosis we irradiated human lung fibroblasts in vitro. We focused on following points: 1. cell growth 2. synthesis of fibronectin 3. synthesis of collagen (procollagen-I-peptid) 4. synthesis of TGF-beta-1 Methods Human fetal lung fibroblasts (MRC-5 ,ICN Biochemicals Eschwege ,Germany) cultured in DME-medium plus 10 % FCS plus L-glutamine, penicillin G, amphotericin B and gentamycine; air humidity 100 %, temp. 37°C, CO2 5%; change of medium twice weekly and 24 hr. before measurements. 24hrs. after seeding, application of ionizing radiation (CO 60; 4.5, 7.5, 10.5 Gy ). Measurements on day 3,6,9,12,15 after irradiation: Fibronectin (ELISA), Takara TGF beta (ELISA), DPC Biermann Procollagen-I-Peptide (ELISA), Takara LDH ( kinetic assay), Sigma Cell counts (counting chamber) All measurements have been done twice. Results 1. cell growth was inhibited in a dose dependent manner. 2. Beginning at day 3 cell related synthesis of fibronectin was increased depending on the dose of irradiation. 3. Similar observations were made in synthesis of procollagen-I-peptide. 4. TGF-beta levels were increased four fold after irradiation beginning on day 6 and returned to basal values between day 9 and 15 (the cells treated with 10.5 Gy were an exception. Here we found a furthermore higher secretion rate ). 5. No elevation of LDH was noticed, showing that cytolysis was not important in these effects. Discussion Irradiation of fetal human lung fibroblasts inihibited cell growth in a dose depend manner. This was not due to cell death initiated by ionizing rays, because LDH ( marker of cytolysis) was not elevated in culture supernatants. We assume that irradiation induces differentiation of progenitor-fibroblasts to promitotic fibrocytes as reported by other groups. TGF-beta was considerably elevated in culture supernatants after irradiation. TGF-beta is assumed to play a key role in fibrosing disease of lung, liver and kidney and may be involved in radiation induced lung fibrosis as well. Our experiments show, that fibroblasts are able to produce high amounts of TGF-beta and fibronectins - even if inflammatory cells are absent- and may stimulate themselves in an autocrine manner.This mechanism is thought to be an important co-factor in the pathobiology of different fibrosing disorders of the lung and may be important in radiation injury of the lung as well. Conclusion Fibroblasts produce increased amounts of TGF-beta and fibronectin after irradiation. They may play a more active role in the pathogenesis of radiation injury than thought up to now.

Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 34-36).

<p>Heparan sulfate (HS) is a polysaccharide of glycosaminoglycan type composed of alternating hexuronic acid [either glucuronic acid (GlcA) or iduronic acid (IdoA)] and glucosamine (GlcN) units that can be sulfated in various positions. HS binds to a large number of proteins and these interactions promote many biological processes, including cell adhesion and growth factor signaling. This thesis deals with the structural analysis of short heparan sulfate sequences that mediate binding to fibroblast growth factors FGF1 and FGF2, their receptor FGFR4, and the angiogenesis inhibitor endostatin.</p><p>Both FGF1 and FGF2 were shown to interact with N-sulfated hexa- and octasaccharide fragments isolated from HS. A pool of HS fragments depleted for FGF1 binding retained the ability to bind FGF2. Changes in 6-O sulfation affected binding to FGF1 but not FGF2, indicating that these proteins bind to distinct HS sequences. </p><p>All octasaccharides with high affinity for FGF1 contained an internal IdoA2S-GlcNS6S-IdoA2S trisaccharide motif as shown by exoenzyme-based sequence analysis. FGF2 bound to a mono-O-sulfated hexasaccharide with an internal IdoA2S unit, although the affinity was higher for a di-O-sulfated octasaccharide displaying an IdoA2S-GlcNS-IdoA2S trisaccharide motif. </p><p>FGFR4 was shown to bind the HS analogue heparin with a K_D value of 0.3 μM.</p><p>The interaction between FGFR4 and HS depends on both IdoA2S and GlcNS6S units. Sequence analysis suggested that the number but not the precise location of 6-O-sulfate groups determines affinity.</p><p>The HS-binding site of endostatin was identified through alanine scanning. Endostatin mutants with reduced affinity for HS were unable to counteract angiogenesis induced by FGF2. The predominant HS motif recognized by endostatin was shown to consist of two N-sulfated domains separated by N-acetylglucosamine units.</p>

<p>In the healing of cutaneous wounds, paracrine communication between keratinocytes and fibroblasts regulates cell differentiation, proliferation and synthesis of extracellular matrix. Deficient epidermal coverage, as seen in burn-wounds, frequently results in hypertrophic scars. Previous studies suggest that keratinocytes downregulate the production of collagen and profibrotic factors in fibroblasts. We hypothesized that keratinocytes downregulate the expression of the profibrotic factor connective tissue growth factor (CTGF) in fibroblasts, and regulate fibroblast expression of genes important to wound healing. In keratinocyte-fibroblast cocultures, keratinocytes downregulated CTGF mRNA and protein in fibroblasts, through the secretion of interleukin-1 (IL-1) α. Using Affymetrix DNA microarrays, it was demonstrated that factors from keratinocytes regulate the expression of 69 genes important to wound healing. The regulation of 16 of these genes was confirmed by Northern blotting, and IL-1α from keratinocytes regulated all the 16 genes examined. IL-1-mediated CTGF gene regulation was further investigated. Both IL-1 isoforms, α and β, suppressed CTGF expression through an inhibition of CTGF promoter activity. Interestingly, transforming growth factor-β-stimulated Smad phosphorylation was not affected by IL-1. Finally, we hypothesized that CTGF is downregulated in burn wound by split-thickness skin grafting and that the expression of CTGF is suppressed during reepithelialization. The expression of CTGF protein was decreased in successfully skin-grafted wound areas, and increased in open, granulating burn wounds. Moreover, CTGF protein expression was absent beneath the migrating edge of reepithelialization <i>ex vivo</i>. In conclusion, we demonstrate that, in <i>in vitro</i> models, keratinocyte-derived IL-1α regulates the expression of CTGF and other genes with importance to wound healing. Furthermore, it is shown that CTGF expression is suppressed by epidermal wound coverage i burn wounds. These findings may have implications for the understanding of keratinocyte-fibroblast interplay during wound healing and in hypertrophic scar pathogenesis.</p>

<p>Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4)disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of <i>N</i>-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified <i>N</i>-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs).</p><p>Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn²⁺-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups.</p><p>The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.</p>

Planar supported, polymerized phospholipid bilayers (PPBs) composed of 1,2-bis[10-(2’,4’-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC or BSPC) were generated by a redox polymerization method. The PPBs were supported by a silicon substrate. The PPBs were characterized and tested for uniformity and stability under physiological conditions. The PPBs were analyzed in vitro with murine derived cells that are pertinent to the host response. Cellular attachment and phenotypic changes in RAW 264.7 macrophages and NIH 3T3 fibroblasts were investigated on PPBs and compared to bare silicon controls. Fluorescent and SEM images were used to observe cellular attachment and changes in cellular behavior. The PPBs showed much lower cellular adhesion for both cell lines than bare silicon controls. Of the cells that attached to the PPBs, a very low percentage showed the same morphological expressions as seen on the controls. The hypothesis generated from this work is that defects in the PPBs mediated the cellular attachment and morphological changes that were observed. Finally, a layer-by-layer (LbL) deposition of a poly(acrylic acid) (PAA) and poly(N-vinylpyrrolidone) (PNVP) alternating bilayer was attempted as a proof of concept for future modification of this system.