Stimulation of hair regrowth in an animal model of androgenic alopecia using 2-deoxy-D-ribose.

Androgenic alopecia (AGA) affects both men and women worldwide. New blood vessel formation can restore blood supply and stimulate the hair regrowth cycle. Recently, our group reported that 2-deoxy-D-ribose (2dDR) is 80%-90% as effective as VEGF in the stimulation of neovascularization in in vitro models and in a chick bioassay. In this study, we aimed to assess the effect of 2dDR on hair growth. We prepared an alginate gel containing 2dDR, polypropylene glycol, and phenoxyethanol. AGA was developed in C57BL6 mice by intraperitoneally injecting testosterone (TE). A dihydrotestosterone (DHT)-treated group was used as a negative control, a minoxidil group was used as a positive control, and we included groups treated with 2dDR gel and a combination of 2dDR and minoxidil. Each treatment was applied for 20 days. Both groups treated with 2dDR gel and minoxidil stimulated the morphogenesis of hair follicles. H&E-stained skin sections of C57BL/6 mice demonstrated an increase in length, diameter, hair follicle density, anagen/telogen ratio, diameter of hair follicles, area of the hair bulb covered in melanin, and an increase in the number of blood vessels. Masson's trichrome staining showed an increase in the area of the hair bulb covered in melanin. The effects of the FDA-approved drug (minoxidil) on hair growth were similar to those of 2dDR (80%-90%). No significant benefit were observed by applying a combination of minoxidil with 2dDR. We conclude that 2dDR gel has potential for the treatment of androgenic alopecia and possibly other alopecia conditions where stimulation of hair regrowth is desirable, such as after chemotherapy. The mechanism of activity of 2dDR remains to be established.

An alginate-Based tube gel delivering 2-deoxy-D-ribose for stimulation of wound healing.

Developing multifunctional wound dressings capable of inducing rapid angiogenesis and with antibacterial activity would be attractive for diabetic and superficial wound healing. Hydrogels delivered from tubes have several desirable features -they are easy to apply, keep the wound moist, reduce the entry of microorganisms and avoid the need for painful dressing removal. Previously we reported that 2 deoxy-D-ribose (2dDR) delivered from a variety of dressings is capable of promoting wound healing by stimulating angiogenesis. Alginate hydrogels are an ideal vehicle to deliver a bioactive agent capable of promoting wound healing. In this study we developed and evaluated a tube hydrogel capable of delivering 2dDR with the aim of achieving a stable, convenient to administer and biologically effective wound treatment. Further, we included the stabilizer 2-phenoxy ethanol which provided antimicrobial activity. We synthesized hydrogels by the Green method, using simple mixing of sodium alginate, propylene glycol, 2-phenoxy ethanol and 2dDR in water. FTIR (Fourier transformation infrared spectroscopy) analysis confirmed an absence of undesirable chemical changes in the gel components, and SEM images of the freeze-dried gels showed porous structures. When 2dDR alginate gel (2dDR-SA hydrogel) was placed in PBS at 37°C, almost 92% of 2dDR was released within 7 days. When tested on cultured cells, 2dDR-SA hydrogels did not inhibit metabolic activity or proliferation, achieving up to 90 and 98% of control respectively over 7 days. 2dDR-SA hydrogel also showed anti-bacterial activity against E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, and MRSA which was attributable to the stabilizer 2-phenoxy ethanol in the hydrogel. Stimulation of angiogenesis in the chorioallantoic membrane assay by 2dDR-SA hydrogel was found to be significant compared to the blank-SA. Wound healing potential was studied in full-thickness wounds in rats where acceleration of wound healing was seen. H&E staining of the wound tissue showed an enhanced number of blood vessels and re-epithelization, and a reduced number of inflammatory cells in 2dDR-SA treated animals compared to blank-hydrogels while Masson's trichrome staining showed increased collagen deposition. In summary we describe a convenient to apply hydrogel which has promise for use in a range of superficial skin wounds including applications in chronic wound care.

Developing Wound Dressings Using 2-deoxy-D-Ribose to Induce Angiogenesis as a Backdoor Route for Stimulating the Production of Vascular Endothelial Growth Factor.

2-deoxy-D-Ribose (2dDR) was first identified in 1930 in the structure of DNA and discovered as a degradation product of it later when the enzyme thymidine phosphorylase breaks down thymidine into thymine. In 2017, our research group explored the development of wound dressings based on the delivery of this sugar to induce angiogenesis in chronic wounds. In this review, we will survey the small volume of conflicting literature on this and related sugars, some of which are reported to be anti-angiogenic. We review the evidence of 2dDR having the ability to stimulate a range of pro-angiogenic activities in vitro and in a chick pro-angiogenic bioassay and to stimulate new blood vessel formation and wound healing in normal and diabetic rat models. The biological actions of 2dDR were found to be 80 to 100% as effective as VEGF in addition to upregulating the production of VEGF. We then demonstrated the uptake and delivery of the sugar from a range of experimental and commercial dressings. In conclusion, its pro-angiogenic properties combined with its improved stability on storage compared to VEGF, its low cost, and ease of incorporation into a range of established wound dressings make 2dDR an attractive alternative to VEGF for wound dressing development.

2-deoxy-d-ribose (2dDR) upregulates vascular endothelial growth factor (VEGF) and stimulates angiogenesis.

BACKGROUND: Delayed neovascularisation of tissue-engineered (TE) complex constructs is a major challenge that causes their failure post-implantation. Although significant progress has been made in the field of angiogenesis, ensuring rapid neovascularisation still remains a challenge. The use of pro-angiogenic agents is an effective approach to promote angiogenesis, and vascular endothelial growth factor (VEGF) has been widely studied both at the biological and molecular levels and is recognised as a key stimulator of angiogenesis. However, the exogenous use of VEGF in an uncontrolled manner has been shown to result in leaky, permeable and haemorrhagic vessels. Thus, researchers have been actively seeking alternative agents to upregulate VEGF production rather than exogenous use of VEGF in TE systems. We have previously revealed the potential of 2-deoxy-d-ribose (2dDR) as an alternative pro-angiogenic agent to induce angiogenesis and accelerates wound healing. However, to date, there is not any clear evidence on whether 2dDR influences the angiogenic cascade that involves VEGF. METHODS: In this study, we explored the angiogenic properties of 2dDR either by its direct application to human aortic endothelial cells (HAECs) or when released from commercially available alginate dressings and demonstrated that when 2dDR promotes angiogenesis, it also increases the VEGF production of HAECs. RESULTS: The VEGF quantification results suggested that VEGF production by HAECs was increased with 2dDR treatment but not with other sugars, including 2-deoxy-l-ribose (2dLR) and d-glucose (DG). The stability studies demonstrated that approximately 40-50% of the 2dDR had disappeared in the media over 14 days, either in the presence or absence of HAECs, and the reduction was higher when cells were present. The concentration of VEGF in the media also fell after day 4 associated with the reduction in 2dDR. CONCLUSION: This study suggests that 2dDR (but not other sugars tested in this study) stimulates angiogenesis by increasing the production of VEGF. We conclude 2dDR appears to be a practical and effective indirect route to upregulating VEGF for several days, leading to increased angiogenesis.

Identification of a fibrin concentration that promotes skin cell outgrowth from skin explants onto a synthetic dermal substitute.

BACKGROUND: Our overall objective is to develop a single-stage in-theatre skin replacement by combining small explants of skin with a synthetic biodegradable dermal scaffold. The aim of the current study is to determine the concentration of fibrin constituents and their handling properties for both adhering skin explants to the scaffold and encouraging cellular outgrowth to achieve reepithelialization. METHODS: Small skin explants were combined with several concentrations of thrombin (2.5,4.5,and 6.5 I.U) and fibrinogen (18.75,67, and 86.5 mg/ml), cultured in Green's media for 14 days and cellular outgrowth was measured using Rose Bengal staining. They were also cultured on electrospun scaffolds for 14 and 21 days. Hematoxylin and eosin (H&E) staining was undertaken to visualize the interface between skin explants and scaffolds and metabolic activity and collagen production were assessed. RESULTS: A thrombin/fibrinogen combination of 2.5 I. U/ml /18.75 mg/ml showed significantly greater cell viability as assessed by Rose Bengal stained areas at days 7 and 14. This was also seen in DAPI images and H&E stains skin explant/scaffold constructs. Fibrin with a concentration of thrombin 2.5 I.U./ml took 5-6 min to set, which is convenient for distributing skin explants on the scaffold. CONCLUSION: The study identified concentrations of thrombin (2.5 I.U/ml) and fibrinogen (18.75 mg/ml), which were easy to handle and aided the retention of skin explants and permitted cell outgrowth from explants.

Multifunctional Copper-Containing Mesoporous Glass Nanoparticles as Antibacterial and Proangiogenic Agents for Chronic Wounds.

The physiological wound healing process involves a cascade of events which could be affected by several factors resulting in chronic, non-healing wounds. The latter represent a great burden especially when bacterial biofilms are formed. The rise in antibiotic resistance amongst infectious microorganisms leads to the need of novel approaches to treat this clinical issue. In this context, the use of advanced biomaterials, which can enhance the physiological expression and secretion of the growth factors involved in the wound healing process, is gaining increasing attention as a robust and appealing alternative approach. Among them, mesoporous glasses are of particular interest due to their excellent textural properties and to the possibility of incorporating and releasing specific therapeutic species, such as metallic ions. One of the most attractive therapeutic ions is copper thanks to its proangiogenic and antibacterial effects. In this contribution, copper containing mesoporous glass nanoparticles were proposed as a multifunctional device to treat chronic wounds. The developed nanoparticles evidenced a very high specific surface area (740 m2/g), uniform pores of 4 nm and an almost total release of the therapeutic ion within 72 h of soaking. The produced nanoparticles were biocompatible and, when tested against Gram positive and Gram negative bacterial species, demonstrated antibacterial activity against both planktonic and biofilm bacteria in 2D cell monolayers, and in a 3D human model of infected skin. Their proangiogenic effect was tested with both the aortic ring and the chick chorioallantoic membrane assays and an increase in endothelial cell outgrowth at a concentration range between 30 and 300 ng/mL was shown. Overall, in this study biocompatible, multifunctional Cu-containing mesoporous glass nanoparticles were successfully produced and demonstrated to exert both antibacterial and proangiogenic effects.

Spatiotemporal release of VEGF from biodegradable polylactic-co-glycolic acid microspheres induces angiogenesis in chick chorionic allantoic membrane assay.

While vascular endothelial growth factor (VEGF) is an acknowledged potent pro-angiogenic agent there is a need to deliver it at an appropriate concentration for several days to achieve angiogenesis. The aim of this study was to produce microspheres of biodegradable polylactic-co-glycolic acid (PLGA) tailored to achieve sustained release of VEGF at an appropriate concentration over seven days, avoiding excessive unregulated release of VEGF that has been associated with the formation of leaky blood vessels. Several formulations were examined to produce microspheres loaded with both human serum albumin (HSA) and VEGF to achieve release of VEGF between 3 and 10 ng per ml for seven days to match the therapeutic window desired for angiogenesis. In vitro experiments showed an increase in endothelial cell proliferation in response to microspheres bearing VEGF. Similarly, when microspheres containing VEGF were added to the chorionic membrane of fertilised chicken eggs, there was an increase in the development of blood vessels over seven days in response, which was significant for microspheres bearing VEGF and HSA, but not VEGF alone. There was an increase in both blood vessel density and branching - both signs of proangiogenic activity. Further, there was clearly migration of cells to the VEGF loaded microspheres. In summary, we describe the development of an injectable delivery vehicle to achieve spatiotemporal release of physiologically relevant levels of VEGF for several days and demonstrate the angiogenic response to this. We propose that such a treatment vehicle would be suitable for the treatment of ischemic tissue or wounds.

Visualisation of the insertion of a membrane for the treatment of preterm rupture of fetal membranes using a synthetic model of a pregnant uterus.

Preterm premature rupture of fetal membranes is a leading cause of preterm delivery. Preterm labour can compromise fetal survival, and even if a pregnancy affected by preterm premature rupture of fetal membrane continues, major complications associated with leakage of amniotic fluid and risk of infection can affect the normal development and survival of the baby. There are limited management options for preterm premature rupture of fetal membrane other than delivery of the baby if ascending infection (chorioamnionitis) is suspected. We have previously reported the development and characterisation of an implantable membrane with the aim of using it to occlude the internal os of the cervix, in order to prevent amniotic fluid loss, allow fluid reaccumulation and reduce the risk of chorioamnionitis. For this, an electrospun biocompatible and distensible bilayer membrane was designed with mechanical properties similar to the human amniotic membrane. In this study, we consider the effects of sterilization on the membrane, how to insert the membrane and visualise it using routine clinical methods. To do this, we used e-beam sterilisation and examined the ability of the membrane to adhere to ex vivo human cervical tissues. We also studied its insertion into a custom-synthesised model of a 20-week pregnant uterus and imaged the membrane using ultrasound. Sterilisation produced minor effects on physical and mechanical properties, but these did not affect the capacity of the membrane to be sutured or to provide a fluid barrier. We demonstrated that fibrin glue can successfully adhere the bilayer membrane to cervical tissues. Finally, we demonstrated that the membrane can be inserted through the cervix as well as visualized in place using ultrasound imaging and an endoscope. In summary, we suggest this membrane is a candidate for further development in an appropriate animal model, supported by appropriate imaging, to precede possible future human studies if judged to demonstrate satisfactory safety and efficacy profiles.

Developing improved tissue-engineered buccal mucosa grafts for urethral reconstruction.

INTRODUCTION: We aimed to compare alternative synthetic scaffolds suitable for future implantation and to examine the use of an inhibitor of lysyl oxidase (beta-amino-propionitrile [ß-APN]) to reduce contraction in these implants. METHODS: Three synthetic scaffolds were compared to natural dermis as substrates for the production of tissue-engineered skin. For natural dermis, Euroskin was used to provide a cell-free cadaveric dermis. Synthetic scaffolds consisted of microfibrous poly-L-lactic acid (PLA), nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and a micro-/nanofibrous trilayer of PLA-PHBV-PLA. The latter were all electrospun and then all four scaffolds (three synthetic, one natural) were placed in six well plates. A culture well was formed on the scaffold using a 1 cm diameter stainless steel ring and 1.5×105 oral fibroblasts were seeded one side; after two days of culture, the ring was placed on the other side of the scaffolds and 3×105 oral keratinocytes were seeded on to the scaffolds and cultured with keratinocytes uppermost. After a further two days of culture, scaffolds were cut to 1 cm2 and raised to an air-liquid interface on stainless steel grids; some were treated with 200 µg/mL ß-APN throughout the culture period (28 days). Contraction in vitro was assessed by serial digital photography of cell-seeded scaffolds and cell-free scaffolds three times a week for 28 days. All cell-seeded scaffolds were assessed for cell metabolic activity, mechanical properties, histology, and morphology by scanning electron microscopy (SEM). RESULTS: The mean fibre diameters and pore sizes of PLA and PHBV scaffolds were 2.4±0.77, 0.85±0.21 µm (p<0.001), and 10.8±2.3, 4.3±1.1 µm (p<0.001), respectively. Oral fibroblasts and keratinocytes were tightly adhered and grew well on both surfaces of trilayer. The ultimate tensile strength (UTS) and Young's modulus (YM) of PLA samples were significantly lower than Euroskin (p<0.001 and p<0.05, respectively); only the UTS of the trilayer samples was slightly significantly lower (p<0.05). Metabolic activity was significantly increased for cells on all scaffolds, without significant differences between them from Day 0 to Day 28. There were no adverse effects of ß-APN on cell viability. With respect to contraction, cells on trilayer and PHBV monolayers did not undergo any significant contraction; however, cells on PLA monolayer and Euroskin contracted 25.3% and 56.4%, respectively, over 28 days. The addition of 200 µg/ml ß-APN significantly reduced contraction of Euroskin compared with the control (p<0.01); however, ß-APN did not affect PLA contraction during this culture period (p>0.05). CONCLUSIONS: This study shows that a trilayer micro-nano-3D porous synthetic scaffold is suitable for oral keratinocyte and fibroblast growth with good cell viability and minimal contraction. This material also has good mechanical properties and histological analyses showed its ability to mimic normal human oral mucosal morphology. Furthermore, synthetic trilayer scaffolds have advantages over biological scaffolds - there is no risk of disease transmission or immunological rejection and they appear resistant to contraction. We suggest they present a good alternative to allodermis for future use in urethral reconstruction.

Developing Repair Materials for Stress Urinary Incontinence to Withstand Dynamic Distension.

BACKGROUND: Polypropylene mesh used as a mid-urethral sling is associated with severe clinical complications in a significant minority of patients. Current in vitro mechanical testing shows that polypropylene responds inadequately to mechanical distension and is also poor at supporting cell proliferation. AIMS AND OBJECTIVES: Our objective therefore is to produce materials with more appropriate mechanical properties for use as a sling material but which can also support cell integration. METHODS: Scaffolds of two polyurethanes (PU), poly-L-lactic acid (PLA) and co-polymers of the two were produced by electrospinning. Mechanical properties of materials were assessed and compared to polypropylene. The interaction of adipose derived stem cells (ADSC) with the scaffolds was also assessed. Uniaxial tensiometry of scaffolds was performed before and after seven days of cyclical distension. Cell penetration (using DAPI and a fluorescent red cell tracker dye), viability (AlamarBlue assay) and total collagen production (Sirius red assay) were measured for ADSC cultured on scaffolds. RESULTS: Polypropylene was stronger than polyurethanes and PLA. However, polypropylene mesh deformed plastically after 7 days of sustained cyclical distention, while polyurethanes maintained their elasticity. Scaffolds of PU containing PLA were weaker and stiffer than PU or polypropylene but were significantly better than PU scaffolds alone at supporting ADSC. CONCLUSIONS: Therefore, prolonged mechanical distension in vitro causes polypropylene to fail. Materials with more appropriate mechanical properties for use as sling materials can be produced using PU. Combining PLA with PU greatly improves interaction of cells with this material.

Development of an implantable synthetic membrane for the treatment of preterm premature rupture of fetal membranes.

Preterm premature rupture of fetal membranes is a very common condition leading to premature labour of a non viable fetus. Significant morbidities may occur when preterm premature rupture of fetal membranes management is attempted to prolong the pregnancy for fetal maturation. Reducing the rate of loss of amniotic fluid and providing a barrier to bacterial entry may allow the pregnancy to continue to term, avoiding complications. Our aim is to develop a synthetic biocompatible membrane to form a distensible barrier for cervical closure which acts to reduce fluid loss and provide a surface for epithelial ingrowth to help repair the damaged membranes. Therefore, a bilayer membrane was developed using an electrospinning technique of combining two FDA-approved polymers, poly-L-lactic acid (PLA) and polyurethane (Z3) polymer. This was compared to a plain electrospun Z3 membrane. The physical and mechanical properties were assessed using scanning electron microscope images and a BOSE tensiometer, respectively, and compared to native fetal membranes. The performance of the membranes in preventing fluid loss was assessed by measuring their ability to support a column of water. Finally the ability of the membranes to support cell ingrowth was assessed by culturing adipose-derived stem cells on the membranes for two weeks and assessing metabolic activity after 7 and 14 days. The physical properties of the bilayer were similar to that of the native fetal membranes and it was resistant to fluid penetration. This bilayer membrane presented mechanical properties close to those for fetal membranes and showed elastic distention, which may be crucial for progress of the pregnancy. The membrane was also able to retain surgical sutures. In addition, it also supported the attachment and growth of adipose-derived stem cells for two weeks. In conclusion, this membrane may prove a useful approach in the treatment of preterm premature rupture of fetal membranes and now merits further investigation.

Production of ascorbic acid releasing biomaterials for pelvic floor repair.

OBJECTIVE: An underlying abnormality in collagen turnover is implied in the occurrence of complications and recurrences after mesh augmented pelvic floor repair surgeries. Ascorbic acid is a potent stimulant of collagen synthesis. The aim of this study is to produce ascorbic acid releasing poly-lactic acid (PLA) scaffolds and evaluate them for their effects on extracellular matrix production and the strength of the materials. MATERIALS AND METHODS: Scaffolds which contained either l-ascorbic acid (AA) and Ascorbate-2-Phosphate (A2P) were produced with emulsion electrospinning. The release of both drugs was measured by UV spectrophotometry. Human dermal fibroblasts were seeded on scaffolds and cultured for 2weeks. Cell attachment, viability and total collagen production were evaluated as well as mechanical properties. RESULTS: No significant differences were observed between AA, A2P, Vehicle and PLA scaffolds in terms of fibre diameter and pore size. The encapsulation efficiency and successful release of both AA and A2P were demonstrated. Both AA and A2P containing scaffolds were significantly more hydrophilic and stronger in both dry and wet states compared to PLA scaffolds. Fibroblasts produced more collagen on scaffolds containing either AA or A2P compared to cells grown on control scaffolds. CONCLUSION: This study is the first to directly compare the two ascorbic acid derivatives in a tissue engineered scaffold and shows that both AA and A2P releasing electrospun PLA scaffolds increased collagen production of fibroblasts to similar extents but AA scaffolds seemed to be more hydrophilic and stronger compared to A2P scaffolds. STATEMENT OF SIGNIFICANCE: Mesh augmented surgical repair of the pelvic floor currently relies on non-degradable materials which results in severe complications in some patients. There is an unmet and urgent need for better pelvic floor repair materials. Our current understanding suggests that the ideal material should be able to better integrate into sites of implantation both biologically and mechanically. The impact of vitamin C on extracellular matrix production is well established but we in this study have undertaken a critical comparison of two derivatives of vitamin C as they are released from a biodegradable scaffold. This strategy proved to be equally useful with both derivatives in terms of new tissue production yet we observed significant differences in mechanical properties of these biomaterials.

Biomaterials for pelvic floor reconstructive surgery: how can we do better?

Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are major health issues that detrimentally impact the quality of life of millions of women worldwide. Surgical repair is an effective and durable treatment for both conditions. Over the past two decades there has been a trend to enforce or reinforce repairs with synthetic and biological materials. The determinants of surgical outcome are many, encompassing the physical and mechanical properties of the material used, and individual immune responses, as well surgical and constitutional factors. Of the current biomaterials in use none represents an ideal. Biomaterials that induce limited inflammatory response followed by constructive remodelling appear to have more long term success than biomaterials that induce chronic inflammation, fibrosis and encapsulation. In this review we draw upon published animal and human studies to characterize the changes biomaterials undergo after implantation and the typical host responses, placing these in the context of clinical outcomes.

The effect of ascorbic acid and fluid flow stimulation on the mechanical properties of a tissue engineered pelvic floor repair material.

Synthetic non-degradable meshes used in pelvic floor surgery can cause serious complications such as tissue erosion. A repair material composed of an autologous oral fibroblast seeded degradable polylactic acid scaffold may be a viable alternative. The aims of this study were to investigate the effects of media supplementation with additives (ascorbic acid-2-phosphate, glycolic acid and 17-ß-oestradiol) on the mechanical properties of these scaffolds. Oral fibroblasts were isolated from buccal mucosa. The effects of the three additives were initially compared in two-dimensional culture to select the most promising collagen stimulating additive. Sterile electrospun scaffolds were seeded with 500,000 oral fibroblasts and fixed in 6-well plates and subjected to ascorbic acid-2-phosphate (the best performing additive) and/or mechanical stimulation. Mechanical stimulation by fluid shear stress was induced by rocking scaffolds on a platform shaker for 1 h/day for 10 of 14 days of culture. In two-dimensional culture, ascorbic acid-2-phosphate (concentrations from 0.02 mM to 0.04 M) and glycolic acid (10 µM) led to significantly greater total collagen production, but ascorbic acid-2-phosphate at 0.03 mM produced the greatest stimulation (of the order of >100%). In three-dimensional culture, mechanical stimulation alone gave non-significant increases in stiffness and strength. Ascorbic acid-2-phosphate (0.03 mM) significantly increased collagen production in the order 280% in both static and mechanically stimulated scaffolds (p < 0.0001). There was no additional effect of mechanical stimulation. Dense collagen I fibres were observed with ascorbic acid-2-phosphate supplementation. Uniaxial tensiometry showed that strength (p < 0.01) and stiffness (p <0.05) both improved significantly. A combination of ascorbic acid-2-phosphate and mechanical stimulation led to further non-signficant increases in strength and stiffness. In conclusion, a pelvic floor repair material with improved mechanical properties can be developed by supplementing culture media with ascorbic acid-2-phosphate to increase collagen I production. Future studies will assess the change in mechanical properties after implantation in an animal model.

Acute in vivo response to an alternative implant for urogynecology.

PURPOSE: To investigate in vivo the acute host response to an alternative implant designed for the treatment of stress urinary incontinence (SUI) and pelvic organ prolapse (POP). METHODS: A biodegradable scaffold was produced from poly-L-lactic acid (PLA) using the electrospinning technique. Human and rat adipose-derived stem cells (ADSCs) were isolated and characterized by fluorescence-activated cell sorting and differentiation assays. PLA scaffolds were seeded and cultured for 2 weeks with human or rat ADSCs. Scaffolds with and without human or rat ADSCs were implanted subcutaneously on the abdominal wall of rats. After 3 and 7 days, 6 animals from each group were sacrificed. Sections from each sample were analyzed by Haematoxylin and Eosin staining, Sirius red staining, and immunohistochemistry for CD68, PECAM-1, and collagen I and III. RESULTS: Animals responded to the scaffolds with an acute macrophage response. After 7 days of implantation, there was extensive host cell penetration, new blood vessel formation, and new collagen deposition throughout the full thickness of the samples without obvious differences between cell-containing and cell-free scaffolds. CONCLUSIONS: The acute in vivo response to an alternative implant (both with and without cells) for the treatment of SUI and POP showed good acute integration into the host tissues.

Developing a tissue engineered repair material for treatment of stress urinary incontinence and pelvic organ prolapse-which cell source?

AIMS: Synthetic non-absorbable meshes are widely used to augment surgical repair of stress urinary incontinence (SUI) and pelvic organ prolapse (POP); however, there is growing concern such meshes are associated with serious complications. This study compares the potential of two autologous cell sources for attachment and extra-cellular matrix (ECM) production on a biodegradable scaffold to develop tissue engineered repair material (TERM). METHODS: Human oral fibroblasts (OF) and human adipose-derived stem cells (ADSC) were isolated and cultured on thermo-annealed poly-L-lactic acid (PLA) scaffolds for two weeks under either unrestrained conditions or restrained (either with or without intermittent stress) conditions. Samples were tested for cell metabolic activity (AlamarBlue® assay), contraction (serial photographs analyzed with image J software), total collagen production (Sirius red assay), and production of ECM components (immunostaining for collagen I, III, and elastin; and scanning electron microscopy) and biomechanical properties (BOSE tensiometer). Differences were statistically tested using two sample t-test. RESULTS: Both cells showed good attachment and proliferation on scaffolds. Unrestrained scaffolds with ADSC produced more total collagen and a denser homogenous ECM than OF under same conditions. Restrained conditions (both with and without intermittent stress) gave similar total collagen production, but improved elastin production for both cells, particularly OF. The addition of any cell onto scaffolds led to an increase in biomechanical properties of scaffolds compared to unseeded scaffolds. CONCLUSIONS: OF and ADSC both appear to be suitable cell types to combine with biodegradable scaffolds, in the development of a TERM for the treatment of SUI and POP. Neurourol. Urodynam. 33:531-537, 2014. © 2013 Wiley Periodicals, Inc.

Tissue engineering airway mucosa: a systematic review.

OBJECTIVES/HYPOTHESIS: Effective treatments for hollow organ stenosis, scarring, or agenesis are suboptimal or lacking. Tissue-engineered implants may provide a solution, but those performed to date are limited by poor mucosalization after transplantation. We aimed to perform a systematic review of the literature on tissue-engineered airway mucosa. Our objectives were to assess the success of this technology and its potential application to airway regenerative medicine and to determine the direction of future research to maximize its therapeutic and commercial potential. DATA SOURCES AND REVIEW METHODS: A systematic review of the literature was performed searching Medline (January 1996) and Embase (January 1980) using search terms "tissue engineering" or "tissue" and "engineering" or "tissue engineered" and "mucous membrane" or "mucous" and "membrane" or "mucosa." Original studies utilizing tissue engineering to regenerate airway mucosa within the trachea or the main bronchi in animal models or human studies were included. RESULTS: A total of 719 papers matched the search criteria, with 17 fulfilling the entry criteria. Of these 17, four investigated mucosal engineering in humans, with the remaining 13 studies investigating mucosal engineering in animal models. The review demonstrated how an intact mucosal layer protects against infection and suggests a role for fibroblasts in facilitating epithelial regeneration in vitro. A range of scaffold materials were used, but no single material was clearly superior to the others. CONCLUSION: The review highlights gaps in the literature and recommends key directions for future research such as epithelial tracking and the role of the extracellular environment.

Glucomannan-poly(N-vinyl pyrrolidinone) bicomponent hydrogels for wound healing.

Polysaccharides interact with cells in ways that can be conducive to wound healing. We have recently reported that konjac glucomannan (KGM) which is comprised of d-mannose and d-glucose linked by ß-1,4 glycosidic chains, stimulates fibroblast proliferation. The aim of this study was to produce a range of crosslinked KGMs and bicomponent KGM containing hydrogels and to examine their potential for wound healing. Two types of KGM hydrogel were synthesized, biodegradable from crosslinked KGM and non-biodegradable by forming semi-IPNs and graft-conetworks with a second synthetic component, poly(N-vinyl pyrrolidinone-co-poly(ethyleneglycol)diacrylate) (P(NVP-co-PEGDA)), which was produced by UV initiated radical polymerization. Crosslinked KGM was formed by bimolecular termination of macro-radicals formed by oxidation with Ce(iv). Semi-IPNs were formed by copolymerization of NVP and PEGDA in the presence of KGM and in the graft-conetworks the KGM was also crosslinked using the Ce(iv) procedure. The hydrogels had different swelling properties and differences could be observed in their chemical structure using 13C solid state NMR, DSC and FTIR. Both forms were cytocompatible but only the graft-conetworks had the ability to stimulate fibroblast metabolic activity and to stimulate the migration of both fibroblasts and keratinocytes. In conclusion a form of KGM hydrogel has been produced that could benefit wound healing.

Comparison of candidate scaffolds for tissue engineering for stress urinary incontinence and pelvic organ prolapse repair.

OBJECTIVES: To identify candidate materials which have sufficient potential to be taken forward for an in vivo tissue-engineering approach to restoring the tissue structure of the pelvic floor in women with stress urinary incontinence (SUI) or pelvic organ prolapse (POP). MATERIALS AND METHODS: Oral mucosal fibroblasts were seeded onto seven different scaffold materials, AlloDerm ( LifeCell Corp., Branchburg, NJ, USA), cadaveric dermis, porcine dermis, polypropylene, sheep forestomach, porcine small intestinal submucosa (SIS) and thermoannealed poly(L) lactic acid (PLA) under both free and restrained conditions. The scaffolds were assessed for: cell attachment using AlamarBlue and 4,6-diamidino-2-phenylindole (DAPI); contraction using serial photographs; and extracellular matrix production using Sirius red staining, immunostaining and scanning electron microscopy. Finally the biomechanical properties of all the scaffolds were assessed. RESULTS: Of the seven, there were two biodegradable scaffolds, synthetic PLA and natural SIS, which supported good cell attachment and proliferation. Immunostaining confirmed the presence of collagen I, III and elastin which was highest in SIS and PLA. The mechanical properties of PLA were closest to native tissue with an ultimate tensile strength of 0.72 ± 0.18 MPa, ultimate tensile strain 0.53 ± 0.16 and Young's modulus 4.5 ± 2.9 MPa. Scaffold restraint did not have a significant impact on the above properties in the best scaffolds. CONCLUSION: These data support both PLA and SIS as good candidate materials for use in making a tissue-engineered repair material for SUI or POP.

High molecular weight plant heteropolysaccharides stimulate fibroblasts but inhibit keratinocytes.

Konjac glucomannan (KGM) is a natural polysaccharide of ß(1-4)-D-glucomannopyranosyl backbone of D-mannose and D-glucose derived from the tuber of Amorphophallus konjac C. Koch. KGM has been reported to have a wide range of activities including wound healing. In this study we examined KGM extracts prepared from five plant species, (Amorphophallus konjac Koch, Amorphophallus oncophyllus, Amorphophallus prainii, Amorphophallus paeoniifolius and Amorphophallus elegans) for their effects on cultured human keratinocytes and fibroblasts. Extracts from A. konjac Koch, A. oncophyllus and A. prainii (but not from A. paeoniifolius or A. elegans) stimulated fibroblast proliferation both in the absence and presence of serum. However, these materials inhibited keratinocyte proliferation. The fibroblast stimulatory activity was associated with high molecular weight fractions of KGM and was lost following ethanol extraction or enzyme digestion with ß-mannanase. It was also reduced by the addition of concanavalin A but not mannose suggesting that these heteropolysaccharides are acting on lectins but not via receptors specific to mannose. The most dramatic effect of KGM was seen in its ability to support fibroblasts for 3weeks under conditions of deliberate media starvation. This effect did not extend to supporting keratinocytes under conditions of media starvation but KGM did significantly help support adipose derived stem cells under media starvation conditions.