Developing a synthetic composite membrane for cleft palate repair.

An oronasal fistula is a passage between the oral and nasal cavity. Currently, surgical procedures use mucosal flaps or collagen grafts to make a barrier between oral and nasal cavities. Our aim was to develop a cell-free synthetic repair material for closure of nasal fistulas. We surface functionalized electrospun polyurethane (PU) and poly-L-lactic acid (PLLA) and composite polymer (PU-PLLA) membranes with acrylic acid through plasma polymerization. Membranes were treated in a layer-by-layer approach to develop highly charged electrostatic layer that could bind heparin as a pro-angiogenic glycosaminoglycan. The properties were evaluated through physical, chemical, and mechanical characterization techniques. Cytotoxicity was tested with MC3T3 pre-osteoblast cell lines for 3, 7, and 14 days, and vasculogenesis was assessed by implantation into the chorio-allantoic membrane in chick embryos for 7 days. In vivo biocompatibility was assessed by subcutaneous implantation in rats for 1, 3, and 6 weeks. The membranes consisted of random fibers of PLLA-PU with fiber diameters of 0.47 and 0.12 µm, respectively. Significantly higher cell proliferation and migration of MC3T3 cells at 3, 7, and 14 days were shown on plasma-coated membranes compared with uncoated membranes. Further, it was found that plasma-coated membranes were more angiogenic than controls. In vivo implantation of membranes in rats did not reveal any gross toxicity to the materials, and wound healing was comparable with the native tissue repair (sham group). We therefore present a plasma-functionalized electrospun composite polymer membrane for use in the treatment of fistulas. These membranes are flexible, non-cytotoxic, and angiogenic, and we hope it should lead to permanent closure of oronasal fistula.

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.

Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis.

Porous microspheres have the potential for use as injectable bone fillers to obviate the need for open surgery. Successful bone fillers must be able to support vascularisation since tissue engineering scaffolds often cease functioning soon after implantation due to a failure to vascularise rapidly. Here, we test the angiogenic potential of a tissue engineered bone filler based on a photocurable acrylate-based high internal phase emulsion (HIPE). Highly porous microspheres were fabricated via two processes, which were compared. One was taken forward and investigated for its ability to support human mesenchymal progenitor cells and angiogenesis in a chorioallantoic membrane (CAM) assay. Porous microspheres with either a narrow or broad size distribution were prepared via a T-junction microfluidic device or by a controlled stirred-tank reactor of the HIPE water in oil in water (w/o/w), respectively. Culture of human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells showed proliferation over 11 days and formation of cell-microsphere aggregates. In-vitro, hES-MP cells were found to migrate into microspheres through their surface pores over time. The presence of osteoblasts, differentiated from the hES-MP cells, was evidenced through the presence of collagen and calcium after 30 days. Microspheres pre-cultured with cells were implanted into CAM for 7 days and compared with control microspheres without pre-cultured cells. The hES-MP seeded microspheres supported greater angiogenesis, as measured by the number of blood vessels and bifurcations, while the empty scaffolds attracted host chick cell ingrowth. This investigation shows that controlled fabrication of porous microspheres has the potential to create an angiogenic, bone filling material for use as a cell delivery vehicle.

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.

Simple surface coating of electrospun poly-L-lactic acid scaffolds to induce angiogenesis.

Tissue-engineered constructs often fail due to poor integration with the patient's tissues. Specifically, they fail to be neovascularised, leading to the death and loss of the implanted tissues. Thus, there is a need to produce angiogenic materials to improve tissue integration. We describe the development of a layer-by-layer approach to coat electrospun scaffolds to help promote angiogenesis into these biomaterials once implanted. Electrospun poly-L-lactic acid was coated comparing two different techniques - one using alternative layers of polyethyleneImine (PEI) and polyacrylic Acid (PAC) and one with alternative layers of PEI and heparin for a total of seven layers in both cases. Both scaffolds were then coated with heparin as the final layer. The scaffold coated with alternate PEI and PAC showed a clear ability to bind the most heparin. This scaffold was then studied further for its ability to bind vascular endothelial growth factor, which was confirmed using an ELISA. The scaffold coated with seven alternate layers of PEI and PAC and heparin was then implanted in a chick chorionic allantoic membrane (CAM) assay. After a period of 7 days in the CAM, the coated scaffold showed strong angiogenic activity. In contrast, the uncoated scaffolds did not promote angiogenesis. We conclude that this approach to functionalising scaffolds is effective within a clinically relevant time period (7 days in an in-vivo angiogenic model) and suggest this will be useful for improving integration of scaffolds once implanted.