Tissue engineered buccal mucosa for urethroplasty: progress and future directions.

PURPOSE: Autologous buccal mucosa is commonly utilized in the surgical treatment of urethral strictures. Extensive strictures require a larger quantity of tissue, which may lead to donor site morbidity. This review assesses progress in producing tissue engineered buccal mucosa as an alternative graft material. RESULTS: Few clinical studies have introduced cells onto biological or synthetic scaffolds and implanted resulting constructs in patients. The available studies show that buccal mucosa cells on acellular human dermis or on collagen matrix lead to good acute stage tissue integration. Urothelial cells on a synthetic substrate also perform well. However while some patients do well many years post-grafting, others develop stricture recurrence. Acellular biomaterials used to treat long urethral defects in animals commonly lead to fibrosis. CONCLUSIONS: Tissue engineered buccal mucosa shows promise as a substitute for native tissue. The fibrosis which occurs months post-implantation may reflect the underlying disease process recurring in these patients.

Application of layer-by-layer coatings to tissue scaffolds - development of an angiogenic biomaterial.

Tissue engineered materials aimed at wound care typically underperform due to poor engrafting to the wound bed. The need for such materials will continue to intensify as a result of an ageing population and an increase in patients suffering from vascular problems. Here we describe the development of an angiogenic coating strategy employing a combination of plasma phase deposition of acrylic acid and layer-by-layer (LBL) chemistry using polyethyleneimine and poly(acrylic acid) for the immobilization of heparin and Vascular Endothelial Growth Factor (VEGF). The formation of the coating and its ability to immobilize heparin was examined by Quartz Crystal Microbalance with Dissipation. X-ray Photoelectron Spectroscopy (XPS) and Atomic Force Microscopy were used to confirm that these coatings retained a significant amount of heparin on the surface when applied to a flat substrate. The coating strategy was transferred to 2 different tissue scaffold architectures: a commercially available non-biodegradable polypropylene mesh, and a biodegradable electrospun poly(lactic-co-glycolic acid) (PLGA) scaffold. XPS confirmed that the coating was successfully applied to the scaffolds and that a similar amount of heparin was immobilized. In vitro testing showed that while HDMEC readily attached to the PLGA scaffold, they were inhibited from adhering and forming proliferative colonies where heparin alone was attached to the LBL coated PLGA scaffold. However, after dip coating with VEGF, the heparin coated scaffold supported both attachment and colony growth of HDMEC; no such colony formation occurred in the absence of VEGF.

Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.

Development of a calcium-chelating hydrogel for treatment of superficial burns and scalds.

AIMS: Superficial burns and scalds are usually managed conservatively with traditional dressings. Failure to heal within 3 weeks leads to their management by skin grafting. Our aim was to develop a biomaterial to actively promote keratinocyte migration in superficial burns by modulating local cation concentrations to accelerate keratinocyte migration and deter wounds from contracting, thus potentially reducing the number of such wounds requiring grafting. MATERIALS & METHODS: We investigated polymeric hydrogels for their Ca(2+) chelating properties and enhancement of keratinocyte migration in human tissue-engineered skin models. RESULTS: Dimethylaminoethyl methacrylate:methacrylic acid hydrogel coupled with elevated [Mg(2+)] reduced media [Ca(2+)], potentiating keratinocyte migration in tissue-engineered skin models, it also significantly reduced wound model contraction. CONCLUSION: Dimethylaminoethyl methacrylate:methacrylic acid hydrogels could promote wound healing and reduce wound contraction, a significant complication in burn wound healing.

Co-culture of intestinal epithelial and stromal cells in 3D collagen-based environments.

AIM: To investigate the co-culture of established intestinal epithelial cell lines and stromal cells in a series of collagen-based environments for production of tissue-engineered intestinal epithelium for in vitro investigations. MATERIALS & METHODS: Intestinal epithelial cells were co-cultured with fibroblasts on a range of supporting collagen matrices including commercially available Promogran and on collagen-based gels. RESULTS: Epithelial growth was achieved with one combination of vimentin-expressing stromal and cytokeratin-expressing intestinal epithelial cells grown on collagen gels supplemented with Matrigel, and held at an air-liquid interface. CONCLUSIONS: Collagen-based gels can support the co-culture of intestinal epithelial and stromal cells resulting in the growth of an epithelium that has some morphological similarity to normal intestinal tissue.

Transglutaminase inhibitors induce hyperproliferation and parakeratosis in tissue-engineered skin.

BACKGROUND: The transglutaminase (TG) family consists of eight distinct isoforms. TG types 1, 3 and 5 play a major role in normal skin development, with TG2 also being elevated during dermal wounding. TG1, 3 and 5 are responsible for the cross-linking of keratin precursors and formation of the cornified envelope during keratinocyte differentiation. TG2 may play a role in keratinocyte basement membrane cross-linking. Abnormal TG expression has been demonstrated in Darier disease, Netherton syndrome, psoriasis and lamellar ichthyosis. During a recent investigation of skin contraction in tissue-engineered skin, transglutaminase inhibitors were found to produce hyperproliferation and parakeratosis. OBJECTIVES: Accordingly, this study was designed to study the effect of pan-transglutaminase inhibition on morphology of tissue-engineered skin and expression of keratinocyte differentiation and proliferation-associated antigens. METHODS: We used a tissue-engineered model of human skin, based on de-epidermized acellular human dermis, seeded with normal keratinocytes and dermal fibroblasts and cultured at an air-liquid interface. The pan-transglutaminase inhibitors putrescine, NTU283 (1-dimethyl,2-[(oxopropyl)thio]imidazolium) and NTU285 (N-benzyloxycarbonyl-l-glutaminyl-6-dimethylsulfonium-5-oxo-l-norleucine) were added to the culture medium. After 28 days, histology and immunohistochemistry for collagen IV, involucrin and cytokeratins 6, 10 and 16 were performed. RESULTS: Keratinocyte hyperproliferation and parakeratosis were seen in response to transglutaminase inhibition. Inhibition of transglutaminase also resulted in loss of basement membrane collagen IV. Involucrin and cytokeratins 6 and 16 were confined to the basal layers in control composites but expressed throughout the epidermis in response to transglutaminase inhibition. A distinct band of expression of cytokeratin 10 was seen in the upper stratum granulosum of control composites but only patchy expression was seen after transglutaminase expression. CONCLUSIONS: Pan-transglutaminase inhibition inhibits terminal differentiation of keratinocytes, leading to a hyperproliferative epidermis with parakeratosis and enhanced expression of involucrin and cytokeratins 6 and 16. Expression of the differentiation-associated cytokeratin, cytokeratin 10, is reduced. Basement membrane integrity is also lost as a result of transglutaminase inhibition.

Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model.

BACKGROUND: Hypertrophic scarring and skin graft contracture are major causes of morbidity after burn injuries. A prominent feature is excessive fibroplasia with accumulation of increased fibrillar collagen relative to normal scar tissue. The application of split-thickness skin grafts or cultured epithelial autografts to burn wounds is known to reduce scarring and contraction. OBJECTIVES: To investigate further how the keratinocyte influences underlying fibroblast behaviour by examining the influence of keratinocytes on fibroblast collagen synthesis, using a new assay for collagen synthesis never previously applied to skin cell biology. METHODS: We investigated the influence of the keratinocyte on fibroblast synthesis of type I collagen using an immunoassay for the aminoterminal propeptide of type I collagen (P1NP) in conditioned medium from monocultures and cocultures of keratinocytes and fibroblasts over 14 days. The importance of the physical presence of the keratinocyte was investigated by comparing cocultures of keratinocytes and fibroblasts against fibroblast monocultures with keratinocyte-conditioned medium. Pharmacological agents known to promote fibroblast proliferation [basic fibroblast growth factor (bFGF)], keratinocyte proliferation [insulin-like growth factor (IGF)-1], modify scarring in vivo[tumour necrosis factor (TNF)-alpha] or modify collagen biochemistry [putrescine, estrone, estradiol and beta-aminopropionitrile (beta-APN)] were then investigated for their effect on collagen synthesis in fibroblasts and in keratinocyte/fibroblast cocultures. RESULTS: Keratinocytes in coculture with fibroblasts, and keratinocyte-conditioned medium, both reduced fibroblast P1NP synthesis. Of the pharmacological agents investigated, bFGF, IGF-1, TNF-alpha and beta-APN all increased collagen synthesis both in monocultures of fibroblasts and in cocultures of keratinocytes and fibroblasts. CONCLUSIONS: Fibroblast collagen synthesis appears to be downregulated by keratinocyte-derived cytokines. Fibroblast growth factors and proinflammatory cytokines appear to be able partially to overcome this downregulation and to increase collagen synthesis.

Tissue-engineered buccal mucosa for substitution urethroplasty.

OBJECTIVE: To develop tissue-engineered buccal mucosa (TEBM) for use in substitution urethroplasty, as urethral reconstruction is limited by the amount and type of tissue that is available for grafting, and BM has become the favoured tissue for use as a urethral substitute in the last decade. MATERIALS AND METHODS: After enzymatic treatment of a small (0.5 cm) BM biopsy the epidermis and dermis were mechanically separated. Oral keratinocytes were isolated from the epidermis and oral fibroblasts from the dermis. These cells were expanded and applied to sterilized de-epidermized dermis (DED) to obtain a full-thickness TE oral mucosa. Horizontal migration of keratinocytes on the DED was assessed using a tetrazolium-blue (MTT) assay. The TEBM was assessed histologically after mechanical stressing in vitro using catheterization and meshing. RESULTS: Histologically the TEBM closely resembled the native oral mucosa after culturing at an air-liquid interface for 2 weeks. The MTT assay showed good horizontal migration of keratinocytes on the DED. Serial histology revealed a gradually increasing thickness of the epidermis and remodelling of the dermis by the fibroblasts from day 1 to day 14. Despite subjecting the TEBM to mechanical stress the integrity of the epidermal-dermal junction was maintained. CONCLUSIONS: We report the successful culture of full-thickness TEBM for substitution urethroplasty, which is robust and suitable for clinical use.

Developmental and species differences in the response of the ureter to metabolic inhibition.

The effect of inhibiting oxidative phosphorylation on electrically stimulated phasic and high-K+ depolarization-induced tonic contractions in ureteric smooth muscle has been investigated. Intracellular [Ca2+] and pH were monitored fluorimetrically with simultaneous tension measurement, in adult and neonatal rat and guinea-pig ureter. Little difference was found in the response of adult or neonatal rat ureters; cyanide abolished phasic contractions and intracellular Ca2+ transients. The contractions of the adult guinea-pig ureter were also reduced by cyanide, but not as much as those of the adult rat. Neonatal guinea-pig was, however, remarkably resistant to the effects of cyanide, with force and Ca2+ transients remaining at control levels after an initial transient dip. These differences between tissues were not apparent when a high K+ concentration was used to depolarize tissues and produce maintained [Ca2+]i and force changes; cyanide reduced force but not [Ca2+]i in all preparations. Intracellular pH decreased in all preparations with inhibition of oxidative phosphorylation, but this did not correlate with changes in contraction. It is concluded that there are both species and developmental differences in the response to metabolic inhibition of the ureter which lead to differing changes in contractile activity.

Developmental changes in intracellular pH buffering power in smooth muscle.

Intracellular pH (pHi) is known to modulate contraction. Neonatal tissues can differ from adult tissue in contractile response to stimuli known to alter pHi e.g. hypoxia. Changes of pH are attenuated by buffering, thus any difference in buffering power (beta) between tissues could affect their functional response to pHi perturbation. Similarly the extent to which any extracellular pH (pHo) alteration is transmitted into a pHi change will also influence function. We have therefore determined the intrinsic beta and effect of pHo change on pHi in neonatal and adult ureteric, uterine and gastric smooth muscles using the pH-sensitive fluorophore carboxy-SNARF. beta was found to be similar in the three adult tissues, but there were significant differences between neonatal tissues. In contrast, we found little difference in the amount of pHi change produced by pHo change between neonatal and adult tissues from the same smooth muscle, but a difference between smooth muscles. These data highlight significant differences between smooth muscles and their developmental state, which may contribute to different degrees of protection when pH is perturbed.

The effects of metabolic inhibition on force, Ca2+ and pHi in guinea-pig ureteric smooth muscle.

The effect of metabolic inhibition on the contractile function of adult guinea-pig ureter has been investigated. Strips of ureteric smooth muscle were loaded with Indo-1 or SNARF to measure intracellular [Ca2+] ([Ca2+]i) or pH (pHi) simultaneously with force. Inhibiting oxidative phosphorylation with cyanide rapidly reduced phasic contractions and the associated Ca2+ transients, after initial transient increases. The effects of cyanide were reversible and related to the amount of contractile activity undertaken. Inhibition of glycolysis with iodoacetate abolished all force. In high-K+-depolarised preparations, cyanide reduced the tonic contraction, but this was not accompanied by a reduction in [Ca2+]i, suggesting a desensitisation of the myofilaments. Cyanide produced a fall in pHi, which may underlie the initial transient increase in force. These data suggest that metabolic inhibition reduces force in the ureter by affecting both excitation and hence the Ca2+ transient, and at the myofilaments to reduce their sensitivity to Ca2+. Thus when oxidative metabolism is impaired contractile dysfunction may arise in the ureter.

The role of the sarcolemmal Ca(2+)-ATPase in the pH transients associated with contraction in rat smooth muscle.

1. We have investigated the origin of the intracellular acid pH transients that accompany myometrial contraction. Intra- and extracellular pH were measured with SNARF and intracellular Ca2+ concentration ([Ca2+]i) with indo-1. 2. An intracellular acidification accompanied spontaneous contractions and those elicited by KCl depolarization or the addition of the agonists carbachol or prostaglandin F2 alpha. The size of the acidification increased with the magnitude of the contraction. 3. The intracellular acidification was accompanied by an extracellular alkalinization, showing that it results from proton movement across the surface membrane. Furthermore, it was decreased either by addition of Cd2+ (20 nM, an inhibitor of the sarcolemmal Ca(2+)-ATPase) or by elevating [Ca2+]o. 4. Extracellular alkalinization increased the magnitude of the rise of [Ca2+]i and force produced by KCl. 5. An intracellular acidification was also associated with contraction in the portal vein and ureter. 6. We conclude that the sarcolemmal Ca(2+)-ATPase produces a significant intracellular acidification while removing Ca2+. Both the acidification and decrease of [Ca2+]i will promote relaxation. Since Ca2+ and protons have opposite effects on many cellular processes, this dual regulation by these two ions may be of general importance.

Enantiomers of myo-inositol-1,3,4-trisphosphate and myo-inositol-1,4,6 -trisphosphate: stereospecific recognition by cerebellar and platelet myo-inositol-1,4,5-trisphosphate receptors.

The naturally occurring tetrakisphosphate myo-inositol-1,3,4, 6-tetrakisphosphate [Ins(1,3,4,6)P4] was able to release Ca2+ from the intracellular stores of permeabilized rabbit platelets but was 40-fold less potent than D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. The Ca2+ releasing activity of Ins(1,3,4,6)P4 was rationalized by envisaging two alternative receptor binding orientations in which the vicinal D-1,6-bisphosphate of Ins(1,3,4,6)P4 mimics the D-4,5-bisphosphate in the Ins(1,4,5)P3 binding conformation. This rationalization predicted that Ins(1,4,5)P3 regioisomers [i.e, D-myo-inositol -1,4,6-trisphosphate [D-Ins(1,4,6)P3] and D-myo-inositol-1,3,6 -trisphosphate [D-Ins(1,3,6)P3]] should also possess Ca(2+)-releasing activity. The unambiguous total synthesis of the enatiomers of Ins(1,4,6)P3 [i.e., D-Ins(1,4,6)P3 and D-Ins(3,4,6)P3] and the enatiomers of Ins(1,3,4)P3 [i.e., D-Ins(1,3,6)P3 and D-Ins(1,3,4)P3] allowed an examination of this prediction. D-Ins(1,4,6)P3 released Ca2+ from the intracellular stores of permeabilized platelets and was only 2-3-fold less potent than Ins(1,4,5)P3. D-Ins(1,3,6)P3 [alternative nomenclature, L-Ins(1,3,4)P3] also released Ca2+ but was 12-fold less potent than Ins(1,4,5)P3. Both D-Ins(1,4,6)P3 and D-Ins(1,3,6)P3 displaced specifically bound [3H]Ins(1,4,5)P3 from the Ins(1,4,5)P3 receptor on rat cerebellar membranes. In contrast, however, D-Ins(3,4,6)P3 [alternative nomenclature, L-Ins(1,4,6)P3] and D-Ins(1,3,4)P3 neither possessed Ca(2+)-releasing activity nor displaced [3H]Ins(1,4,5)P3. The ability of D-Ins(1,3,6)P3 to release Ca2+ in permeabilized platelets is in contrast to its apparent lack of Ca(2+)-mobilizing activity previously reported in rat basophilic leukemic cells. The possibility that this is a reflection of the different Ins(1,4,5)P3 receptor subtypes possessed by these two cell types is discussed.

A role for protein phosphorylation in modulating Ca2+ elevation in rabbit platelets treated with thapsigargin.

The effect of modifying protein kinase and phosphatase activity on Ca2+ influx induced by inhibition of Ca(2+)-ATPase activity has been investigated in rabbit platelets. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or inhibition of phosphatase type 1/2A (PP1/2A) activity with calyculin A caused a dose-dependent inhibition of cytosolic Ca2+ elevation in thapsigargin (Tg)-treated platelets and decreased Ca2+ influx into platelets at a time when Ca2+ channels had already been opened by pretreatment of cells with Tg. In addition, both activation of PKC and inhibition of PP1/2A activity caused a dose-dependent inhibition of bivalent cation (Mn2+) influx (acting as a surrogate for Ca2+ influx) in Tg-treated platelets. Inhibition of cyclo-oxygenase activity caused a small decrease in [Ca2+]i elevation in Tg-treated platelets, but had no effect on the ability of PMA or calyculin A to inhibit Tg-induced [Ca2+]i elevation Unexpectedly, PMA inhibited Tg-induced [Ca2+]i elevation in the absence of extracellular Ca2+, and in agreement calyculin A decreased [Ca2+]i elevation almost to basal levels. The results from this study were confirmed with another Ca(2+)-ATPase inhibitor, namely 2,5-di(tert-butyl)hydroquinone (tBHQ). These findings therefore suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues plays a role in the regulation of both Ca2+ influx and in the filling state of the intracellular Ca2+ pool in platelets treated with Tg.