In vivo implantation of a tissue engineered stem cell seeded hemi-laryngeal replacement maintains airway, phonation, and swallowing in pigs.

Laryngeal functional impairment relating to swallowing, vocalisation, and respiration can be life changing and devastating for patients. A tissue engineering approach to regenerating vocal folds would represent a significant advantage over current clinical practice. Porcine hemi-larynx were de-cellularised under negative pressure. The resultant acellular scaffold was seeded with human bone marrow derived mesenchymal stem cells and primary human epithelial cells. Seeded scaffolds were implanted orthotopically into a defect created in the thyroid cartilage in 8 pigs and monitored in vivo for 2 months. In vivo assessments consisted of mucosal brushing and bronchoscopy at 1, 2, 4, and 8 weeks post implantation followed by histological evaluation post termination. The implanted graft had no adverse effect on respiratory function in 6 of the 8 pigs; none of the pigs had problems with swallowing or vocalisation. Six out of the 8 animals survived to the planned termination date; 2 animals were terminated due to mild stenosis and deep tissue abscess formation, respectively. Human epithelial cells from mucosal brushings could only be identified at Weeks 1 and 4. The explanted tissue showed complete epithelialisation of the mucosal surface and the development of rudimentary vocal folds. However, there was no evidence of cartilage remodelling at the relatively early censor point. Single stage partial laryngeal replacement is a safe surgical procedure. Replacement with a tissue engineered laryngeal graft as a single procedure is surgically feasible and results in appropriate mucosal coverage and rudimentary vocal fold development.

Characterization of a biologically derived rabbit tracheal scaffold.

There is a clinical need to provide replacement tracheal tissue for the pediatric population affected by congenital defects, as current surgical solutions are not universally applicable. A potential solution is to use tissue engineered scaffold as the framework for regenerating autologous tissue. Rabbit trachea were used and different detergents (Triton x-100 and sodium deoxycholate) and enzymes (DNAse/RNAse) investigated to create a decellularization protocol. Each reagent was initially tested individually and the outcome used to design a combined protocol. At each stage the resultant scaffold was assessed histologically, molecularly for acellularity and matrix preservation. Immunogenicity of the final scaffold was assessed by implantation into a rat model for 4 weeks. Both enzymes and detergents were required to produce a completely acellular (DNA content 42.78 ng/mg) scaffold with preserved collagen and elastin however, GAG content were reduced (8.78 ± 1.35 vs. 5.5 ± 4.8). Following in vivo implantation the scaffold elicited minimal immune response and showed significant cellular infiltration and vasculogenesis. The luminal aspect of the implanted scaffold showed infiltration of host derived cells, which were positive for pan cytokeratin. It is possible to create biologically derived biocompatible scaffolds to address specific pediatric clinical problems. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2126-2135, 2017.

Pilot study of a novel vacuum-assisted method for decellularization of tracheae for clinical tissue engineering applications.

Tissue engineered tracheae have been successfully implanted to treat a small number of patients on compassionate grounds. The treatment has not become mainstream due to the time taken to produce the scaffold and the resultant financial costs. We have developed a method for decellularization (DC) based on vacuum technology, which when combined with an enzyme/detergent protocol significantly reduces the time required to create clinically suitable scaffolds. We have applied this technology to prepare porcine tracheal scaffolds and compared the results to scaffolds produced under normal atmospheric pressures. The principal outcome measures were the reduction in time (9 days to prepare the scaffold) followed by a reduction in residual DNA levels (DC no-vac: 137.8±48.82 ng/mg vs. DC vac 36.83±18.45 ng/mg, p<0.05.). Our approach did not impact on the collagen or glycosaminoglycan content or on the biomechanical properties of the scaffolds. We applied the vacuum technology to human tracheae, which, when implanted in vivo showed no significant adverse immunological response. The addition of a vacuum to a conventional decellularization protocol significantly reduces production time, whilst providing a suitable scaffold. This increases clinical utility and lowers production costs. To our knowledge this is the first time that vacuum assisted decellularization has been explored. Copyright © 2015 John Wiley & Sons, Ltd.

Characterisation of porcine dermis scaffolds decellularised using a novel non-enzymatic method for biomedical applications.

Off-the-shelf availability of tissue-engineered skin constructs, tailored by different combinations of reagents to produce a highly preserved biological matrix is often the only means to help patients suffering skin damage. This study assessed the effect of five different decellularisation methods on porcine dermal scaffolds with regard to matrix composition, biomechanical strength, and cytotoxicity using an in vitro biocompatibility assay. Results demonstrated that four out of the five tested decellularisation protocols were efficient in producing acellular scaffolds. Nevertheless, decellularisation method using osmotic shock without enzymatic digestion showed to be efficient not only in removing cellular material and debris from dermal scaffolds but was also beneficial in the preservation of extracellular matrix components (glycosaminoglycans and collagen). Histological assessment revealed that the dermal architecture of coarse collagen bundles was preserved. Examinations by scanning electron microscopy and transmission electron microscopy showed that the arrangement and ultrastructure of collagen fibrils in the scaffolds were retained following non-enzymatic method of decellularisation and also after collagen crosslinking using genipin. Moreover, this decellularised scaffold was not only shown to be biologically compatible when co-cultured with bone marrow-derived mesenchymal stem cells and fibroblasts, but also stimulated the cells to release trophic factors essential for tissue regeneration.

Full-thickness laparoendoscopic colonic excision in an experimental model.

BACKGROUND: Patients requiring surgery for complex colonic polyps traditionally undergo colectomy, with significant risks. Typically in excess of 10-30 cm of colon is removed at laparoscopic or open surgery lasting over 60 min. This study details the preclinical development of a rapid, minimally invasive, limited full-thickness colonic resection. METHODS: Both survival and non-survival procedures were performed in anaesthetized 70-kg pigs. A simulated colonic polyp was created by endoscopic ink injection with a clearance margin delineated by circumferential placement of mucosal argon plasma coagulation marks. Full-thickness eversion of the bowel was achieved using endoscopically placed anchors and the polyp was excised using a laparoscopic stapler. In survival procedures, pigs were killed under anaesthetic 8 days after surgery. All pigs underwent post-mortem examination. RESULTS: Five procedures were performed (5 pigs). The median (range) procedure duration was 26 (20-31) min, with a specimen diameter of 5.1 (4.5-6.3) cm. The postoperative recovery of survival animals (4 pigs) was uneventful. At post-mortem evaluation the resection sites were well healed with no evidence of stenosis, intra-abdominal infection or inadvertent organ damage. Histological assessment of anastomoses showed mucosal repair and restoration of submucosal continuity. CONCLUSION: Full-thickness localized colonic excision with this technique provides a large specimen with adequate healing in a porcine model.

Evaluation of crosslinked and non-crosslinked biologic prostheses for abdominal hernia repair.

INTRODUCTION: Abdominal wall defects and incisional hernias represent a challenging problem. Currently, several commercially available biologic prostheses are used clinically for hernia repair. We compared the performance and efficacy of two non-crosslinked meshes in ventral hernia repair to two crosslinked prostheses in a rodent model. METHODS: Animals were divided into 12 groups (4 matrix types and 3 termination time-points per matrix). A ventral defect was carefully created and overlapped with the biologic prosthesis. RESULTS: Major complications were seroma induction (3 mesh types), implant extrusion (1 mesh type), severe inflammatory and immune responses (non-crosslinked mesh), fibrosis and mineralisation (3 mesh types). After inflammation resolution, 3 of the matrices tested supported hernia healing but with marked tissue and temporal differences. AlloDerm(®*) and Surgisis Gold™ showed tissue reactivity with the host and a rapid rate of matrix remodelling. Bard CollaMend™(*) Implant proved to be inept for hernia repair under the conditions tested. Permacol™ biological implant integration with host tissue increased over time, supporting hernia healing with strength of tissue, and appears to be a safe prosthetic material for ventral hernia repair based on the results of this rodent study.

Individualization of surgical management for early-stage colonic cancer.

A significant number of patients each year undergo radical segmental colectomy for node-negative colonic cancer in the UK. They are likely to represent 50% of our colonic cancer population in the future due to the implementation of the National Bowel Cancer Screening Programme. Short-term morbidity rates of up to 40% are associated with the current surgical treatment and need serious consideration. In this article, we discuss a combination of two techniques that will allow minimally invasive treatment in an increasing number of patients with early colonic cancer.

The "FLEX" procedure: a new technique for full-thickness laparo-endoscopic excision in the colon.

BACKGROUND AND STUDY AIMS: Colonic lesions unsuitable for endoscopic resection and some early cancers that have been incompletely excised endoscopically, are generally treated by segmental colectomy, even though local excision might be adequate. The aim of this study was to develop a laparo-endoscopic procedure for full-thickness local excision of the colon. METHODS: After detailed planning using story-boarding to discuss each step of the procedure, both nonsurvival and survival experiments were performed in seven 50-kg pigs. The technique used was as follows: a simulated colonic polyp was created by endoscopic ink injection; this was inverted using laparo-endoscopically placed BraceBars passing from the outside to the inside of the colon; after laparoscopic over-sewing of the inversion in two layers, endoscopic full-thickness excision was performed. Pigs were sacrificed immediately (n=3) or between 7 and 9 days after surgery (n=4). RESULTS: The procedure was performed without operative perforation or hemorrhage in a median of 233 min (range 201-245 min), and achieved full-thickness excision in all procedures. Specimen diameter was a median of 2.5 cm (range 2-3 cm). All survival pigs convalesced without complication and, at autopsy, normal luminal diameter was confirmed without any peritoneal reaction, hernia or sepsis. Bursting pressures were a median of 245 mmHg (range 240-260 mmHg), with the site of bursting being in normal colon in all but one pig. CONCLUSIONS: This is a novel technique that permits full-thickness laparo-endoscopic excision (FLEX) of a colonic lesion as an alternative to segmental colectomy.

Prolonged maintenance of neointestine using subcutaneously implanted tubular scaffolds in a rat model.

Tissue-engineered small intestine offers a possible alternative to long-term parenteral nutrition or intestinal transplantation in patients with short bowel syndrome. The aim of this study was to investigate the prolonged development of neointestine grown on subcutaneously implanted scaffolds. Tubular polylactide-coglycolide (PLGA) scaffolds were implanted into adult Lewis rats. Four weeks after scaffold implantation, a suspension of organoid units was delivered to the lumen of each scaffold. Organoid units were manufactured from small intestine harvested from neonatal Lewis rats by partial digestion using collagenase and dispase. Scaffolds were removed at 4, 8, and 12 weeks after organoid unit implantation, processed to paraffin, and sectioned. Hematoxylin and eosin staining demonstrated well-developed and well-differentiated intestinal mucosa and a vascularised submucosa within the scaffolds at 4, 8, and 12 weeks. Appearances were similar to native small intestine. Immunohistochemistry performed using primary antibody against proliferating cell nuclear antigen, a marker for cellular proliferation, demonstrated positively staining cells within the mucosa and submucosa at all time points. In the mucosal layer these positively staining cells were found primarily in the crypts. These findings show that neointestinal mucosa can be maintained for at least 12 weeks on a subcutaneous PLGA scaffold, and the presence of actively proliferating cells at 12 weeks suggests potential for further development beyond this.

Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction.

OBJECTIVE: To evaluate placental morphology in pregnancies complicated by early- and late-onset pre-eclampsia (PET) with and without fetal growth restriction (FGR) using stereological techniques. DESIGN: A total of 69 pregnant women were studied. Twenty women had pregnancies complicated by PET, 17 by FGR and 16 by both PET and FUR; the remaining 16 were from gestational-age-matched controls. Each group was further classified into early onset (<34 weeks) and late onsets (>34 weeks) based on gestational ages. SETTING: NPIMR at Northwick Park and St Marks Hospital. POPULATION: placentae from pregnant women. METHODS: Formalin-fixed, wax-embedded sections stained with anti-CD34 antibodies and counterstained with haematoxylin. MAIN OUTCOME MEASURES: Volumes, surface areas, lengths, diameters and shape factors of the villous tissues and fetal vasculature in the intermediate and terminal villi of all the groups studied. RESULTS: Terminal villi volume and surface area were compromised in early-onset PET cases, late-onset PET had no impact on peripheral villi or vasculature features. The morphology of the vascular and villous subcomponents in the intermediate and terminal villi was significantly influenced by late-onset FGR, whereas early-onset FGR caused a reduction in placental weight. Length estimates were not influenced by PET, FGR or age of onset. Intermediate arteriole shape factor was significantly reduced in late-onset FGR. CONCLUSIONS: Isolated early-onset PET was associated with abnormal placental morphology, but placentas from late-onset PET were morphologically similar to placentas from gestational-age-matched controls, confirming the existence of two subsets of this condition and supporting the hypothesis that late-onset PET is a maternal disorder and not a placental disease.

A pilot study investigating a novel subcutaneously implanted pre-cellularised scaffold for tissue engineering of intestinal mucosa.

Tissue engineering of the small intestine offers an alternative to long-term intravenous nutrition and transplantation in patients with intestinal failure. Initial work, although encouraging, is limited by the volume of neonatal tissue required to produce a small neomucosal cyst. Our novel approach is to implant tubular poly-lactide-co-glycolide (PGLA) foam scaffolds subcutaneously. The aim of this study was to investigate whether these scaffolds would support growth of intestinal neomucosa. PGLA scaffolds were implanted subcutaneously into 8 Lewis rats; after 5 weeks, 'organoid units' were injected into the lumens. Tissue was assessed histologically after harvesting and quantitative immunohistochemistry was performed using antibodies against vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGF-R2), fibroblast growth factor basic (bFGF) and fibroblast growth factor receptor 2 (FGF-R2). At 4 weeks post organoid unit implantation, clearly recognisable mucosa and submucosa was present on the luminal surface of the scaffold. Densities of VEGF and VEGF-R2 positive cells increased with time post organoid unit implantation. This pilot study demonstrates that it is possible to tissue engineer small intestinal neomucosa using subcutaneously implanted PLGA scaffolds. The yield of the process compares favourably to the published literature. Further work is required to optimise the technique.

Pre-eclampsia and fetal growth restriction: how morphometrically different is the placenta?

Both pre-eclampsia (PET) and fetal growth restriction (FGR) pose a heavy burden on fetal and maternal health and may disrupt pregnancy outcome. Using design based stereological techniques, placental vascular and villous morphology were assessed to determine the individual role played by both PET and FGR on placental growth during the third trimester. The following placentas delivered between 25 and 41 weeks of gestation were included into the study; controls (n=16), PET (n=20), FGR (n=17) and PET-FGR (n=16). Each placenta was uniformly randomly sampled and the sampled tissue processed to paraffin. Sections were stained with a CD34 antibody and the following morphometric parameters estimated: volumes, surface areas, length, diameters and the shape factor of the villous (terminal and intermediate) and vascular placental features. For stereologically estimated parameters pure PET had an effect on IVS and terminal villi volume only. FGR alone or when coexisting with PET contributed towards significant reductions in volumetric and surface area terminal villous and vascular features. FGR factors also contributed towards a significant reduction in the lengths of all parameters estimated and in the terminal villi diameter. Additionally, FGR was associated with a significant difference in shape factor indices for both intermediate and terminal villi. This study has shown that PET on its own has limited influence on the placental morphology studied, since the vascular features estimated do not differ stereologically from age matched normal controls. However, placental morphology is different between PET and PET-FGR and between PET-FGR and FGR. PET and FGR may have a cumulative effect on placental villous and vascular morphology as seen in the PET-FGR but there is no synergistic effect. These morphological abnormalities may have major physiological implications in terms of placental function and fetal growth.

Reviewing the use of carbon monoxide-releasing molecules (CO-RMs) in biology: implications in endotoxin-mediated vascular dysfunction.

The inducible stress protein heme oxygenase-1 (HO-1) has been linked to tissue and organ protection against the deleterious actions of many pathological conditions, including endotoxin challenge. Similar protection can be achieved by the main products of heme oxygenase activity, namely bilirubin and carbon monoxide (CO). Since the identification of novel chemical compounds that liberate CO in biological systems (CO-releasing molecules or CO-RMs), our group and others have had access to a convenient and simple pharmacological tool that enables to study the role of CO in physiological functions. This article will review the scientific literature published to date on CO-RMs, with emphasis on the in vitro, ex vivo and in vivo experimental models employed to determine the contribution of CO to cellular mechanisms. In addition, we will report on the effect of heme oxygenase-related substances, such as bilirubin, CORM-3 and hemin, in a model of endotoxin-induced hypotension. Among the three different approaches examined, CORM-3 proved the most effective agent in reducing the fall in blood pressure caused by endotoxin. Furthermore, heme oxygenase-related substances affected the endotoxin-stimulated induction and distribution of hepatic HO-1 and inducible nitric oxide synthase (iNOS). Thus, it emerges that CO-RMs could exert important biological actions in the context of endotoxic-mediated dysfunction.

Analyses of the potential oxygen transfer capability in placentae from infants succumbing to sudden infant death syndrome.

BACKGROUND: Morphometric oxygen diffusive conductance (Dp) was estimated to assess the potential efficiency of oxygen transfer across the materno-fetal interface in placentae obtained from victims of sudden infant death syndrome (SIDS). STUDY DESIGN: SIDS placentae were retrieved from archived storage and classified into normal birth weight (NBW, n=16), or small for gestational age (SGA, n=9) and compared against control placentae (n=40) or SGA (n=24) placentae. A combination of stereological techniques and physiological constants were used to estimate total Dp. RESULTS: SIDS NBW cases showed a crucial reduction in fetal capillary surface area when compared with control placentae. SIDS SGA showed a number of deficiencies in basic volumetric and surface area parameters. Values for total and specific Dp in placentae in both SIDS groups were maintained at levels comparable with control and SGA cases, respectively. CONCLUSION: Since more reductions were observed in SIDS SGA group, this suggests that factors responsible for these reductions maybe associated with SGA rather than being SIDS-specific factors.

Morphometric assessment of the oxygen diffusion conductance in placentae from pregnancies complicated by intra-uterine growth restriction.

The morphometric oxygen diffusive conductance (D(p)) of the placenta provides a measure of the efficiency of oxygen transfer between the mother and the developing fetus. Any change in the D(p)may point towards possible adaptation in the light of altered oxygen transfer. Placentae from normal (n=40) and small for gestational age SGA (n=24) pregnancies were analysed using stereological techniques. Each placenta was uniform randomly sampled and tissue samples processed to wax infiltration and embedding using conventional histological preparatory methods. A combination of stereological techniques and physiological constants were used to estimate the partial conductances across the five major tissue compartments involved in oxygen transfer. There was a significant reduction in both fetal birthweight and placental weight in the SGA group when compared with controls. A decrease in both chorionic (S(cv)) and fetal capillary (S(fc)) surface area was also observed in SGA placentae when compared with controls (P>0.001). Villous membrane harmonic thickness (T(vm)) was reduced in the SGA placentae (2.33 microm) when compared with controls (2.67 microm P=0.019). This resulted in a reduction in the minimum D(p)in SGA placentae when compared with controls (P=0.023). Adjusting for fetal weight resulted in no difference in the specific diffusive conductance. Changes in T(vm)in SGA placentae combined with changes in basic surface areas were insufficient to maintain overall D(p)values comparable with control placentae.

The role of human-derived fibrin sealant in the reduction of postoperative flexor tendon adhesion formation in rabbits.

This study assessed the role of a novel fibrin sealant (Vivostat in adhesion reduction after flexor tendon surgery. The deep flexor tendons of the 2nd and 4th digits of the left paw of 20 rabbits were exposed and a standard partial injury was performed on each. The rabbits were randomized to either immediate post-injury treatment with Vivostat or no treatment. In each case active movement of the 2nd digit was prevented while the 4th digit was allowed to move normally. The two groups were assessed at 14 days for adhesion formation with a tensiometer. The right paw acted as the unoperated control. Results showed that there was no significant difference in the force needed to remove the tendon from its sheath when comparing the two Vivostat((R))-treated groups to the unoperated controls. There was, however, a highly significant difference in this force between the non-Vivostat-treated groups and the unoperated controls. This suggests a beneficial effect of Vivostat in reducing post surgical tendon adhesion formation.

Quantitative neuropathological analysis of sudden infant death syndrome.

Detailed stereological analyses of specific regions of brains of children who had died from Sudden Infant Death Syndrome (SIDS) was undertaken to determine whether global evidence of an underlying pathology exists, contributing to an increased susceptibility to SIDS. A significant reduction in the total number of neocortical neurones and neurone volume was observed in SIDS normal birth weight (NBW) infants in comparison to controls. A significant reduction in both volume and total neurone number were also noted in the dorsal motor nucleus of the vagus in SIDS NBW group when compared with controls. Anomalies in regions of the brain involved with cardiorespiratory control (brainstem) and arousal (brainstem and neocortex) may play a crucial role in the chain of events resulting in a SIDS event.