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Reconstruction of a tendon rupture is surgically challenging as each end of the tendon retracts, leaving a substantial gap and direct repair is often not feasible. A tendon graft is required to bridge this defect and restore function. Presently, these gaps are filled with auto-, allo-, or synthetic grafts, but they all have clinical limitations. To address this issue, we developed tissue-engineered grafts by a rapid process using compressed type I collagen, which is the most dominant protein in the tendon. However, biomechanical properties were found to be unsuitable to withstand complete load-bearing in vivo. Hence, a modified suture technique was previously developed to reduce the load on the engineered collagen graft to aid integration in vivo. Using this technique, we tested engineered collagen grafts in vivo on a lapine model in three groups up to 12 weeks without immobilization. Gross observation at 3 and 12 weeks showed the bridge integrated without adhesions with a significant increase in the mechanical, structural and histological properties as compared to 1 week. Insertion of a tissue-engineered collagen graft using a novel load-bearing suture technique which partially loads in vivo showed integration, greater mechanical strength and no adhesion formation in the time period tested. This collagen graft has inherent advantages as compared to the present-day tendon grafts.

Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularized xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X-100 (Vac-STx), and sodium dodecyl sulfate (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50 ng/mg of tissue, while retaining 85% of the sulfate glycosaminoglycan content, and as such was selected as the optimal methodology for decellularization. The resultant Vac-OS scaffolds (decellularized extracellular matrix [dcECM]) were also devoid of the immunogenic alpha-Gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry and fluorescence lifetime imaging microscopy. The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells, respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2 macrophage-driven regenerative response both at 2 and 8 weeks postimplantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1 macrophage-mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair.

When serious cutaneous injury occurs, the innate wound healing process attempts to restore the skin’s appearance and function. Wound healing outcome is affected by factors such as contraction, revascularisation, regeneration versus fibrosis and re-epithelialisation and is also strongly influenced by the pattern and extent of damage to the dermal layer. Dermal replacement scaffolds have been designed to substitute for lost tissue, provide a structure to promote dermal regeneration, and aid skin grafting, resulting in a superior healing outcome. In this study the wound healing properties of a novel fibrin-alginate dermal scaffold were assessed in the porcine wound healing model and also compared to two widely used dermal scaffolds and grafting alone. The fibrin-alginate scaffold, unlike the other scaffolds tested, is not used in combination with an overlying skin graft. Fibrin scaffold treated wounds showed increased, sustained superficial blood flow and reduced contraction during early healing while showing comparable wound closure, re-epithelialisation and final wound outcome to other treatments. The increase in early wound vascularisation coupled with a decrease in contraction and no requirement for a skin graft suggest that the fibrin-based scaffold could provide an effective, distinctive treatment option to improve healing outcomes in human patients.

Tracheal replacement for the treatment of end-stage airway disease remains an elusive goal. The use of tissue-engineered tracheae in compassionate use cases suggests that such an approach is a viable option. Here, a stem cell-seeded, decellularized tissue-engineered tracheal graft was used on a compassionate basis for a girl with critical tracheal stenosis after conventional reconstructive techniques failed. The graft represents the first cell-seeded tracheal graft manufactured to full good manufacturing practice (GMP) standards. We report important preclinical and clinical data from the case, which ended in the death of the recipient. Early results were encouraging, but an acute event, hypothesized to be an intrathoracic bleed, caused sudden airway obstruction 3 weeks post-transplantation, resulting in her death. We detail the clinical events and identify areas of priority to improve future grafts. In particular, we advocate the use of stents during the first few months post-implantation. The negative outcome of this case highlights the inherent difficulties in clinical translation where preclinical in vivo models cannot replicate complex clinical scenarios that are encountered. The practical difficulties in delivering GMP grafts underscore the need to refine protocols for phase I clinical trials.

The growing number of patients requiring liver transplantation for chronic liver disease cannot be currently met due to a shortage in donor tissue. As such, alternative tissue engineering approaches combining the use of acellular biological scaffolds and different cell populations (hepatic or progenitor) are being explored to augment the demand for functional organs. Our goal was to produce a clinically relevant sized scaffold from a sustainable source within 24 h, while preserving the extracellular matrix (ECM) to facilitate cell repopulation at a later stage. Whole porcine livers underwent perfusion decellularization via the hepatic artery and hepatic portal vein using a combination of saponin, sodium deoxycholate, and deionized water washes resulting in an acellular scaffold with an intact vasculature and preserved ECM. Molecular and immunohistochemical analysis (collagen I and IV and laminin) showed complete removal of any DNA material, together with excellent retention of glycosaminoglycans and collagen. Fourier-transform infrared spectroscopy (FTIR) analysis showed both absence of nuclear material and removal of any detergent residue, which was successfully achieved after additional ethanol gradient washes. Samples of the decellularized scaffold were assessed for cytotoxicity by seeding with porcine adipose-derived mesenchymal stem cells in vitro, these cells over a 10-day period showed attachment and proliferation. Perfusion of the vascular tree with contrast media followed by computed tomography (CT) imaging showed an intact vascular network. In vivo implantation of whole intact nonseeded livers, into a porcine model (as auxiliary graft) showed uniform perfusion macroscopically and histologically. Using this method, it is possible to create an acellular, clinically sized, liver scaffold with intact vasculature in less than 24 h.

BackgroundLoss of substantial volumes of laryngeal tissue after trauma or cancer significantly impairs quality of life. We hypothesised that repair of laryngeal defects with a candidate biomaterial, seeded with mesenchymal stem cells (MSC) and epithelial cells, may offer a therapeutic approach to this unmet need.MethodMoulded porous polyhedral oligomeric silsesquioxane polycarbonate-urea (POSS-PCU) scaffolds were seeded with human-derived MSC and epithelial cells, were implanted orthotopically into a defect created in the thyroid cartilage in eight pigs and monitoredin vivofor 2 months.In vivoassessments were performed at 1, 2, 4 and 8 weeks post implantation. Histology was performed following termination.ResultsImplant operations were uncomplicated. One pig was terminated early (2 weeks post-implantation) following expectoration of its implant. No other mortality or morbidity was observed. Endoscopy showed partial extrusion of implants at two weeks and complete extrusion of all implants by termination.ConclusionsPOSS-PCU moulded laryngeal implants, in the present formulation, are extruded from the site of implantation between two- and eight-weeks post-surgery in pigs. In its present formulation and with the present, one-stage, protocol, this material does not appear to provide a suitable scaffold and vehicle for cells intended for partial laryngeal replacement in pigs.

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.

Background and study aims: Current submucosal dissection devices are technically challenging to use, resulting in long and sometimes incomplete colonic polyp resections. The aim of this feasibility preclinical study was to evaluate a new, multimodality instrument with novel electrocautery properties. Methods: Six female adult pigs underwent colonic submucosal resections. The novel device was used to cut mucosa and submucosa using bipolar radiofrequency (BRF; at 400 KHz), provide hemostasis with microwave coagulation (MWC; at 5.8 GHz), and inject fluid via a retractable needle. The main outcomes measured were safety (histological analysis post-recovery), performance, and time needed to achieve complete resection. Results: A total of 12 consecutive colonic pseudopolyps were completely excised (two per subject) using BRF cutting. The median time to complete resection was 44.3 minutes (SD 8.9). The median defect size was 32.8 mm (SD 4.3). MWC was applied on 37 occasions for pre-coagulation or treatment of bleeding vessels. One microperforation was treated successfully with endoscopic clips. All animals recovered uneventfully during the 28-day survival period. Histology confirmed adequate healing in all postmortem defects. Conclusions: In this preclinical evaluation, the novel multimodality endoscopic device facilitated rapid and safe en bloc resection of colonic pseudopolyps.

This paper presents a new haemostatic device utilising microwave energy at 5.8GHz along with the ability to introduce adrenaline for vasoconstriction or other fluids as required. A radiative tip was designed, simulated and manufactured to provide good match into tissue. The complete device was then tested under preclinical conditions at Northwick Park Institute for Medical Research (NPIMR); the results of which along with histological anaylsis are presented herein.

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

Background 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. Method 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. Results 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 vocalization. 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 epithelialization 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. Conclusion 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.

Patients with laryngeal disorders may have severe morbidity relating to swallowing, vocalization, and respiratory function, for which conventional therapies are suboptimal. A tissue-engineered approach would aim to restore the vocal folds and maintain respiratory function while limiting the extent of scarring in the regenerated tissue. Under Good Laboratory Practice conditions, we decellularized porcine larynges, using detergents and enzymes under negative pressure to produce an acellular scaffold comprising cartilage, muscle, and mucosa. To assess safety and functionality before clinical trials, a decellularized hemilarynx seeded with human bone marrow-derived mesenchymal stem cells and a tissue-engineered oral mucosal sheet was implanted orthotopically into six pigs. The seeded grafts were left in situ for 6 months and assessed using computed tomography imaging, bronchoscopy, and mucosal brushings, together with vocal recording and histological analysis on explantation. The graft caused no adverse respiratory function, nor did it impact swallowing or vocalization. Rudimentary vocal folds covered by contiguous epithelium were easily identifiable. In conclusion, the proposed tissue-engineered approach represents a viable alternative treatment for laryngeal defects.

Permacol™ biological implant is a cross-linked porcine dermal collagen biomaterial that has been successfully used in abdominal wall repair. The purpose of this study is to examine biopsies of the implant after use in abdominal wall hernia repair and demonstrate its histological and immunohistochemical characteristics in vivo. Three male patients, ranging in age from 61 to 76 years, underwent biopsy of the implant and surrounding abdominal wall tissue at 16, 36, and 60 months after implantation. At 16 months, the implant demonstrated excellent tissue integration, cellular and neovascular response, and remodeling. The implant–host tissue interface showed seamless integration with no fibrosis. Tissue ingrowth into the material, new blood vessel formation, and remodeling of the host–tissue interface were observed. At approximately 36 months, organized remodeling of the implant into functional, predominately host tissue was generally seen. At more than 60 months, macroscopic and histological characteristics resembled functional human tissue with immunohistochemical evidence of the residual presence of porcine collagen. Permacol™ implant supports controlled and natural long-term remodeling into normal, functional host tissue.

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. Surgical relevance The number of patients diagnosed with large benign colonic polyps and early node-negative cancer has increased since the introduction of colorectal screening. Patients unsuitable for endoscopic treatment undergo colectomy and are therefore subject to a significant risk of morbidity and death. This article describes a novel fusion of laparoscopic and endoscopic elements as a potential transformational alternative to current treatment. The eversion full-thickness laparoendoscopic excision (eFLEX) technique can be performed in under an hour, producing full-thickness colonic specimens exceeding 5·5 cm in diameter. This technique is suitable for translation into clinical practice as an alternative to hemicolectomy, with the potential for reduced morbidity and cost. Presented to the Annual Meeting of the Association of Surgeons of Great Britain and Ireland, Glasgow, UK, May 2013; published in abstract form as Br J Surg 2013; 100(Suppl 7): 2

Biologically derived scaffolds are becoming viable treatment options for tissue/organ repair and regeneration. A continuing hurdle is the need for a functional blood supply to and from the implanted scaffold. We have addressed this problem by constructing an acellular ileal scaffold with an attached vascular network suitable for implantation and immediate reperfusion with the host's blood. Using a vascular perfusion approach, a segment of porcine ileum up to 30 cm long, together with its attached vasculature, was decellularized as a single entity. The quality of the decellularized scaffold was assessed histologically and using molecular tools. To establish vascular perfusion potentials of the scaffold, a right-sided nephrectomy and end-to-end anastomosis of the decellularized scaffold's vasculature to a renal artery and vein were performed in a pig of similar size to the donor animal. Lengths of ileal scaffold, together with its attached vasculature, were successfully decellularized, with no evidence of intact cells/nuclear material or collagen degradation. The scaffold's decellularized vascular network demonstrated optimum perfusion at 1, 2 and 24 h post-implantation and the mesenteric arcade remained patent throughout the assessment. The 1, 2 and 24 h explanted scaffolds demonstrated signs of cellular attachment, with cells positive for CD68 and CD133 on the vascular luminal aspect. It is possible to decellularize clinically relevant lengths of small intestine, together with the associated vasculature, as a single segment. The functional vascular network may represent a route for recellularization for future regeneration of bowel tissue for patients with short bowel syndrome. Copyright © 2013 John Wiley & Sons, Ltd.

Made available in DSpace on 2018-12-11T17:06:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-10-11 Biocompatibility of two newly developed porcine skin scaffolds was assessed after 3, 14, 21 and 90 days of implantation in rats. Both scaffolds showed absence of cells, preservation of ECM and mechanical properties comparable to non-decellularised skin before implantation. Host cell infiltration was much prominent on both scaffolds when compared to Permacol (surgical control). At day 3, the grafts were surrounded by polymorphonuclear cells, which were replaced by a notable number of IL-6-positive cells at day 14. Simultaneously, the number of pro-inflammatory M1-macrophage was enhanced. Interestingly, a predominant pro-remodeling M2 response, with newly formed vessels, myofibroblasts activation and a shift on the type of collagen expression was sequentially delayed (around 21 days). The gene expression of some trophic factors involved in tissue remodeling was congruent with the cellular events. Our findings suggested that the responsiveness of macrophages after non-crosslinked skin scaffolds implantation seemed to intimately affect various cell responses and molecular events; and this range of mutually reinforcing actions was predictive of a positive tissue remodeling that was essential for the long-standing success of the implants. Furthermore, our study indicates that non-crosslinked biologic scaffold implantation is biocompatible to the host tissue and somehow underlying molecular events involved in tissue repair. Post-Graduation in Structural and Functional Biology Federal University of São Paulo (UNIFESP) Department of Biology Instituto de Biociências Letras e Ciências Exatas São Paulo State University (UNESP) Department of Surgical Research Northwick Park Institute for Medical Research University College London (UCL) Department of Biology Instituto de Biociências Letras e Ciências Exatas São Paulo State University (UNESP)

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

This paper presents the first use of Ku band energy at the spot frequency of 14.5GHz for the treatment of a range of dermatological conditions. Initial in-vivo and ex-vivo results are presented, together with histology and data analysis. The outcome from this work indicates that 14.5GHz energy has a strong potential for future use in treating a number of dermatological conditions, where control of depth in the millimeter range, and uniformity over the area of the heating profile, are of paramount importance.

Background. The authors describe the initial validation of a novel full-thickness laparoendoscopic excision (FLEx) technique for the stomach. Methods. The technique was studied in seven 50-kg pigs. Secure full-thickness excision was ensured by inversion excision target with a 1-cm circumferential margin using laparoendoscopically placed brace bars passed intraluminally from the outside of the stomach, laparoscopic oversewing of the site of inversion, and endoscopic full-thickness excision using a dual scope approach. Pigs were sacrificed either immediately (n = 3) or between 7 and 10 days after surgery (n = 4). Results. The procedure achieved uncomplicated full-thickness excision in every case. Median procedure duration was 227 minutes (range = 210-245 minutes). Median specimen diameter was 5.5 cm (range = 2.5-8 cm). Investigative autopsy confirmed technical sufficiency in all animals. Median site bursting pressure was 130 mm Hg (range = 120-160 mm Hg). Conclusions. The FLEx technique proved useful for excision of small localized lesions of the stomach in this animal study.

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.

The aim of this study was to quantitatively define the development of post surgical adhesions (PSAs) in a well characterized experimental model and identify possible windows of pathogenesis where pharmaceutical intervention may be most effective. PSAs were induced, in an established rabbit uterine horn model, using standardized reproducible injury, in 17 experimental groups, each with 8 experimental sites and these PSAs were sampled from 30 seconds to 42 days post surgery. Using design based, unbiased stereology, mean volumes of PSAs and associated tissue damage and reaction per experimental site were calculated for each sample time point. PSA development followed the normal pattern of wound healing with surrounding adjacent tissue having a profound influence and interaction. There was a direct relationship between volume of damage (initial and subsequent) and the volume of injury tissue generated. In vivo weak fibrinous PSAs were present from 10 min following injury, with tenacious fibrinous PSAs present from 1 h and onwards. PSA development can be classified into two distinct stages: (i) PSA modelling — occurring during the first 16 h, in which maximum rate of PSA construction is achieved; and (ii) PSA remodelling — from 16 h onwards. Considering this, PSA prevention should ideally be initiated immediately post injury to prevent PSA modelling or, alternatively, during PSA modelling.

This study was designed to investigate the morphological and functional status of the substance medullaris (MS) of adrenal glands (AG) in progeny of parents who smoked, in varying combinations and amounts, prior to conception. Rats were divided into groups in which either both parents or the mother only or the father only was exposed to cigarette smoke before and during pregnancy together with control groups who were not exposed to smoke at any time. The smoke exposure was calculated to be that experienced by smokers of so called “light” cigarettes. The progeny of these groups were raised to early adulthood and then the experiment was terminated. The adrenal glands of the progeny were examined for functional and morphological status. There were significant differences between the control group and all other groups–most noticeably the group in which mothers were exposed to smoke before and during pregnancy. Surprisingly there were also differences between controls and the groups where the father only had been exposed to cigarette smoke before pregnancy. This raises a whole new set of questions regarding the effect of smoke on genetic material passed form father to fetus and shows that smoking of even so called “light” cigarettes can cause visible morphological changes in progeny from matings where either or both parents have been exposed to this smoke.

The long-term success of vascular bypass graft surgery is greatly compromised by the formation of intimal hyperplasia. An established porcine model was used to investigate the pathology and mechanism resulting in intimal hyperplasia. The aim of this study was to test the specificity of several commercially available antibodies to characterise intimal hyperplasia morphology and its immunological aspects within a porcine model. After bilateral jugular vein graft into the carotid arteries in 12 pigs, vein grafts were harvested at specific time points post-surgery and fixed in 10% neutral buffered formal saline before being embedded in paraffin wax. Five-micrometer sections were cut and subjected to routine histological and immunohistochemical techniques using the ABC method. The following antibodies were tested: von Willebrand factor (vWF), CD31 markers, smooth muscle actin (α-SMC), tissue factor (TF), CD3, two CD45 markers, CD133, NFκ-β and CD68. Results showed that labelled cells were observed with vWF, α-smooth muscle actin, TF, one CD45 marker, CD3 and CD68 within the porcine model. Sections labelled with CD133 were observed in test tissue but not within the intimal hyperplasia. No positivity was observed with either CD31 markers, one vWF or one CD45 marker. In conclusion, although commercially available antibodies may not necessarily be listed as porcine specific, by using different antigen retrieval techniques, satisfactory results can be achieved.

An anal fistula has a primary track passing from an internal anal or rectal opening to an external opening in the perianal area. Surgery aims to eradicate sepsis whilst preserving faecal continence. Fistulotomy, when all tissue caudal to the primary fistulous track is opened, provides the surest method of cure but may diminish patient continence. An alternative sphincter-preserving procedure is to instill a sealant into the track. An experimental porcine model of fistula-in-ano has been developed in the Surgical Research Department at Northwick Park Institute for Medical Research. This allows histological assessment of fistula tracks after novel, sphincter-preserving surgery and treatments have been applied. Under general anaesthetic, 24 anal fistulae were created and treated, three in each of eight adult Large White/Landrace crossbred pigs. Under the same general anaesthetic, a split skin graft was taken from which to culture fibroblasts for future treatment. All tracks were treated at 4 weeks post-track induction when the tracks were established and very similar in clinical appearance to human tracks. All tracks were prepared for treatment using an instrument designed to remove granulation tissue from the wall of the track. Five control tracks were not infilled but simply had their internal and external openings closed with a Vicryl suture. Nine tracks were treated by infill using an acellular porcine dermal collagen matrix. Ten tracks were treated using a mixture of this matrix and autologous cultured fibroblasts. Histological examination of six tracks was carried out at 2 weeks, nine tracks at 2 months and nine tracks at 3 months. Histological assessment demonstrated persistent fistula tracks in only two fistulae, both of which were control tracks. All treated tracks were closed and cured at all times of examination. When autologous fibroblasts were added to the infill material, cellular integration and vascularization were improved. Using this pre-clinical model, provided fistulous tracks were prepared using a new, in-house developed instrument; treatment with acellular collagen matrix alone healed all tracks. Adding autologous fibroblasts improved the quality of wound healing. A pilot study using this treatment in human fistula patients is in progress.

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.

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) 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.

The aim of this study was to assess the histological and thermal effects of different dissection technologies on tissue around incision and to consider their comparative wound healing profiles. The technologies used were Gyrus PlasmaKnife, monopolar electrode, Coblation wand, and Harmonic Scalpel. Single incisions were made using each device on the internal peritoneal surface at four test sites per porcine subject. The incision sites were assessed to determine the extent of tissue and thermal damage around the incision site in the first animal and the formation of wound cover and degree of inflammation 14 days post-procedure in the second. The PlasmaKnife was easier to handle and more controllable than the other devices and resulted in a precise incision with only localised thermal damage. The other technologies produced either deep lesions extending into the muscle and/or considerable lateral damage across the mesothelial surface. Of all the devices only the PlasmaKnife resulted in compact well-organised wound cover showing vascularisation and neo-collagenesis. The notably better wound healing profile and reduced inflammatory response seen suggest that use of the PlasmaKnife in the clinical setting may lead to reductions in both wound healing times and post-operative pain.

Pregnant rats were fed alcohol at 5 and 1.5 g/kg/day. This regime caused dose-dependent neuro-cognitive changes, biochemical changes in neocortex and changes in the morphology of neocortex and cerebellum of descendants. Comparison of different parameter changes across the groups of animals suggests dose-dependent intra-uterine developmental anomalies with considerable adaptive responses to minimal doses of alcohol consumption by mother during pregnancy.

The aim of this study was to investigate the failure of fibrin sealant treatment for fistula-in-ano in an experimental porcine model and to determine histologic changes associated with the sealant and setons. Three surgically created fistulas were treated by seton drainage in each of eight male pigs. After 26 days, magnetic resonance imaging was performed and setons were removed. Two pigs were killed as controls for stereologic histologic fistula track assessment. In six, fistulas were curetted, and in four the fistulas were treated with fibrin sealant. In these four sealant and two seton pigs, magnetic resonance imaging was repeated a median of 47.5 days after fistula formation. The pigs were killed and stereologic histologic fistula track examination was performed to determine granulation tissue and fistula lumen volumes. These values were compared among control, seton, and sealant groups over time, and related to fistula volumes derived from magnetic resonance imaging. Sealant was not visible microscopically within tracks, although some sections revealed a foreign body–type reaction. On stereologic assessment, granulation tissue volumes were smaller in sealant and seton groups than in controls (median, 88 vs. 187 vs. 453 mm3, respectively; P = 0.002) and decreased over time (median, 408 and 152 mm3 (Day 42) vs. 88 and 75 (Day 53), respectively; P = 0.002). Fistula lumen (P < 0.001), and granulation tissue combined with fistula lumen volumes (P = 0.002) were similarly smaller. Magnetic resonance imaging of fistula intensity was less in the sealant group than in the seton group and controls (mean, 777 vs. 978 vs. 1214 units/mm2, P = 0.003). Magnetic resonance imaging fistula volumes were least in sealant and seton groups vs. controls (P = 0.024), decreasing significantly in the sealant group over time (P = 0.018). No direct relationship was found between imaging and histologic volumes. In an experimental porcine model of anal fistula, granulation tissue was still present, albeit diminished, following track curettage combined with seton or sealant therapy, and was minimal in the sealant group, confirming some benefit from this procedure. Eradication of all longstanding granulation tissue may ensure complete success of fibrin sealant therapy.

PURPOSE: A prospective trial was conducted to establish long-term healing of complex idiopathic anorectal fistula, without extension, after fibrin glue treatment, with clinical assessment and magnetic resonance imaging to determine tract healing. METHODS: Twenty-two patients undergoing glue instillation after fistula curettage and irrigation were followed up for a median of 14 months. Clinical assessment, short tau inversion recovery sequence magnetic resonance imaging, and combined short tau inversion recovery and dynamic contrast-enhanced magnetic resonance imaging were performed at a median of three months postoperatively, and their ability to predict outcome in the presence of early skin healing was determined. RESULTS: Of 22 patients, 19 (86.5 percent) had transsphincteric fistulas, 1 (4.5 percent) had a suprasphincteric fistula, 1 (4.5 percent) had an extrasphincteric fistula, and 1 (4.5 percent) had a rectovaginal fistula. None had clinical or radiologic evidence of secondary extension. Despite skin healing in 17 (77 percent) of 22 patients at a median of 14 days after treatment, only 3 (14 percent) remained healed at 16 months. Magnetic resonance imaging with short tau inversion recovery sequences in combination with dynamic contrast-enhanced magnetic resonance imaging predicted outcome in all 10 assessments (100 percent), compared with short tau inversion recovery sequence alone in 16 (94 percent) of 17 assessments or clinical examination in 12 (71 percent) of 17 (P = 0.02). CONCLUSIONS: The success rate of fibrin glue application for complex anorectal fistulas without extension is 14 percent. Magnetic resonance imaging predicts outcome at an earlier stage than clinical examination.

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 μm) when compared with controls (2.67 μm 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.

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.

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.

Previous research has demonstrated impaired renal development, particularly with respect to glomerular number, in victims of sudden infant death syndrome (SIDS). The present study used stereological principles to estimate the volume of the upper lobe of the right lung, total number of terminal bronchiolar duct endings (TBDE), and gas exchange surface area of this lobe within a group of human infants. The infants were classified according to cause of death (SIDS or non-SIDS), and further subdivided according to birth-weight: normal birth-weight (NBW) or low birth-weight (LBW). The results demonstrated that TBDE density was significantly reduced in SIDS compared to non-SIDS (P = 0.014), but only reduced from non-SIDS NBW values in the SIDS NBW group (P = 0.044). Total TBDE number was significantly reduced in SIDS from non-SIDS (P = 0.001), and was significantly reduced from non-SIDS NBW values in SIDS NBW (P = 0.023). Mean gas exchange surface area per TBDE was significantly increased in SIDS compared to non-SIDS cases (P = 0.049). The results of the present study indicate developmental delay of the lung in SIDS NBW infants who had previously not been considered growth retarded based on their normal body parameters. Pediatr Pulmonol. 2001; 31:339–343. © 2001 Wiley-Liss, Inc.

Organ development may be assessed by estimating the total number of functional units within an organ over time; the potential functional capacity of that organ may be represented by the total number of functional units present in the fully developed, mature organ. Relative development of the lung at birth is essential to provide sufficient oxygenation of body tissues and so maintain ex utero life. Estimation of the number of one type of functional unit of the lung – terminal bronchiolar duct endings – provides important information regarding development of the lung. This investigation used stereological techniques, specifically Cavalieri’s Principle and the “physical disector”, to estimate total number of terminal bronchiolar duct endings in the upper lobe of the right lung of a group of 14 control infants between 0 and 66 weeks post-natal age. Results demonstrate that total terminal bronchiolar duct ending number does not increase significantly over the first 24 weeks of post-natal life in normal infants ( P =0.997). The unbiased, design-based techniques used in this paper confirm previous model-based research that indicates that terminal bronchiolar duct ending development is completed before birth.

In victims of sudden infant death syndrome (SIDS), renal development has been reported to be significantly impaired. In the present study, we used stereological techniques to estimate volume of kidney cortex and total number of glomeruli in a group of human infants. Infants were classified according to cause of death—SIDS or non-SIDS. Cases were further subdivided according to birth weight—normal birth weight (NBW) or low birth weight (LBW) (we were unable to identify any non-SIDS LBW infants for our study). No significant differences were found between NBW and LBW infants (irrespective of cause of death) for cortical volume, glomerular density, or total glomerular number ( p > 0.140). Kidney cortical volume, glomerular density, and total glomerular number were not significantly different between SIDS and non-SIDS infants (p > 0.510). Glomerular number was only significantly less in SIDS infants of LBW (p = 0.032) than in controls according to the Wilcoxon rank sum test; using the Kruskal-Wallis for one-way analysis, no significant difference was found (p > 0.010). These results contrast with those from previous studies, as a reduction in glomerular number was not noted in SIDS NBW infants, and the mean value for the control (non-SIDS NBW) group was significantly reduced ( p < 0.01) from those of previous studies. This indicates that glomerular number reduction is seen in SIDS NBW and non-SIDS NBW cases and is therefore directly associated with growth retardation rather than with SIDS.

A novel modification of the physical disector is described which was used to estimate the total number of terminal bronchiolar duct endings (TBDEs) in human infant lung. TBDEs are closed three-dimensional space curves of complex shape that are inherently difficult to count from histological sections. However, careful consideration of the microanatomy of the terminal duct endings provides us with the opportunity to define a very simple and unbiased counting rule. To apply the rule in practice we also need to determine a suitable disector height. Owing to the complex shape of the TBDE we had no prior knowledge of what disector height would be suitable for counting the TBDE structures. Exhaustive serial sectioning of complete TBDE structures was carried out and showed that any disector height under 90 microm would give unbiased counts. A further empirical study was then undertaken to determine the most efficient disector height. This was found to be 50 micro. The total number of TBDEs in the upper lobe of the right lung of six human infants aged between 13 and 25 weeks was also estimated. The estimates of numerical density obtained with our modification of the physical disector were multiplied by estimates of lung lobe volume obtained using Cavalieri's Principle. The total number of TBDEs in the lobes ranged from 15 323 to 57 768, with a mean of 40 306. The average coefficient of error of the number estimates was 19%, which was deemed precise enough given the biological coefficient of variation between TBDE number of 36%.

Research into the pathogenesis of neonatal necrotizing enterocolitis has been a diverse mixture of clinical case reviews, searches for a single cause, analysis of subjective observations and some designed experimental studies. The experimental research has been hampered by the lack of reproducible and clinically appropriate animal models in which to test hypotheses of pathogenic pathways. Reviewing the models which have been evaluated reveals many design inconsistencies and little in depth assessment of the appropriateness of many of these experimental systems. Many species have been studied as potential, clinically adequate models but often without relevant attention to the physiological differences and the effect of age. The piglet seems to present the most extrapolatable data from an appropriate model system for the study of neonatal necrotizing enterocolitis.

A mouse monoclonal antibody which specifically reacts with putative blood-brain barrier (BBB) competent endothelial cells of rat cerebral capillaries was used to identify barrier competent cells in the central nervous system (CNS). The development of the cerebral capillaries and the BBB was examined and quantified, from day 6 to day 40 postpartum, using immunocytochemical and unbiased stereological techniques. There was a progressive increase in capillary formation postnatally, with collateral branching observed with progressive age. BBB development was confined to individual endothelial cells located at the periphery of the cortex until day 10 postpartum. Antibody binding progressively increased postnatally, contributing 30% of the total capillary surface area by day 20. There was a rapid elevation of reactivity from day 20 to day 40, with a mean of 83% by day 40. The BBB constitutes minimal amounts of brain vascular capillaries before day 10 of life in the rat. There is a slower increase in BBB than in total capillaries between days 10 and 20. There is a reversal of this trend between days 20 and 40.

Biologically derived scaffolds are becoming viable treatment options for tissue/organ repair and regeneration. A continuing hurdle is the need for a functional blood supply to and from the implanted scaffold. We have addressed this problem by constructing an acellular ileal scaffold with an attached vascular network suitable for implantation and immediate reperfusion with the host's blood. Using a vascular perfusion approach, a segment of porcine ileum up to 30 cm long, together with its attached vasculature, was decellularized as a single entity. The quality of the decellularized scaffold was assessed histologically and using molecular tools. To establish vascular perfusion potentials of the scaffold, a right-sided nephrectomy and end-to-end anastomosis of the decellularized scaffold's vasculature to a renal artery and vein were performed in a pig of similar size to the donor animal. Lengths of ileal scaffold, together with its attached vasculature, were successfully decellularized, with no evidence of intact cells/nuclear material or collagen degradation. The scaffold's decellularized vascular network demonstrated optimum perfusion at 1, 2 and 24 h post-implantation and the mesenteric arcade remained patent throughout the assessment. The 1, 2 and 24 h explanted scaffolds demonstrated signs of cellular attachment, with cells positive for CD68 and CD133 on the vascular luminal aspect. It is possible to decellularize clinically relevant lengths of small intestine, together with the associated vasculature, as a single segment. The functional vascular network may represent a route for recellularization for future regeneration of bowel tissue for patients with short bowel syndrome.

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.

One of the main problems in healthcare is the loss of tissues resulting from diseases, post-surgery complications or trauma. As a result there is a need for biomaterials designed to promote tissue regeneration and improve wound healing. This study assessed the effect of crosslinking of a porcine dermal collagen matrix with regard to strength of implant/host tissue integration, implant biocompatibility and general healing in a rodent model. Permacol™, a crosslinked acellular collagenous biomaterial was compared with its noncrosslinked equivalent at 3, 6, and 12 months postsubcutaneous implantation. Both matrices were well tolerated and showed no evidence of inflammation or adverse responses either in the host tissue or implants. Progressive integration of the implants with the surrounding tissue was observed. Cellular response was similar for both collagenous matrices although, at 3 and 6 months, noncrosslinked implants showed a significantly higher level of cellular penetration than crosslinked implants. However, at 12 months crosslinked implants showed significantly higher levels of cellular density, neo-vascularisation and integration with host tissue. Additionally, at long term, noncrosslinked implants lost volume suggesting some absorption. The crosslinking process does not seem to be detrimental to cellular response and biocompatibility.

Background Accelerated intimal hyperplasia (IH) is an important cause of morbidity and mortality in patients with atherosclerotic vascular disease treated with bypass vein grafts. We used an interposition vein graft model to determine the source of neointimal cells in a clinically relevant large animal model. Methods Jugular vein segments from sex-mismatched, MHC-in-bred pigs were implanted into common carotid arteries bilaterally and harvested up to 8 weeks postsurgery for stereological, histological, and immunofluorescence analyses. Results Progressive IH lesions contained macrophages and smooth muscle cells (SMC). Fluorescent in situ hybridization following grafting of female veins into male arteries revealed that only ∼10% of the SMC were male, confirming that the majority of intimal SMC derived from the local vessel wall. Conclusions The majority of neointimal SMC in the IH seen after interposition vein grafting derive from the engrafted local vessel wall. These are the first results from a clinically relevant large animal model that confirm data from rodent models. They have implications for the utility of therapeutic stem cells in this type of intimal hyperplasia.

Normal blood flow was measured in two regions of the ileum (distal and proximal) of normal birth weight (NBW) and low birth weight (LBW) neonatal piglets. Compensatory collateral blood flow in response to occlusion of vessels in the mesenteric vascular arcades was also measured in distal and proximal ileum of NBW and LBW neonatal piglets. Under normal control conditions the blood flow in the distal ileum of NBW piglets is reduced (40% less than proximal) and in LBW animals this reduction is greater (55% less than proximal). Both proximal and distal ileum of NBW animals but the proximal ileum only of LBW animals could mount compensatory collateral flow, whereas the distal ileum of LBW animals was unable to do so. This decompensation in LBW distal ileum may explain the predilection of NEC lesions for the distal ileum and proximal colon.

Occlusion of groups of vessels in the mesenteric vascular arcades of distal ileum for 48 h induced necrotizing enterocolitis lesions in low birth weight, spontaneously delivered, neonatal piglets. Lesion severity increased with numbers of adjacent groups of vessels occluded and with proximity to the ileocecal junction. A previously undescribed feature, "prepneumatosis," was confined to the lymphatic vessels of the submucosa and serosa. This feature closely resembled the position, shape, and distribution of classical pneumatosis intestinalis. Occlusion of vessels for 30 min followed by reperfusion did not induce any detectable changes 48 h later. Identical procedures (48-h occlusions) did not induce any detectable changes in 35-kg pigs.

This work investigated the effect of adding nanoparticulate (29 nm) bioactive glass particles on the bioactivity, degradation and in vitro cytocompatibility of poly(3-hydroxybutyrate) (P(3HB)) composites/nano-sized bioactive glass (n-BG). Two different concentrations (10 and 20 wt %) of nanoscale bioactive glass particles of 45S5 Bioglass composition were used to prepare composite films. Several techniques (Raman spectroscopy, scanning electron microscopy, atomic force microscopy, energy dispersive X-ray) were used to monitor their surface and bioreactivity over a 45-day period of immersion in simulated body fluid (SBF). All results suggested the P(3HB)/n-BG composites to be highly bioactive, confirmed by the formation of hydroxyapatite on material surfaces upon immersion in SBF. The weight loss and water uptake were found to increase on increasing bioactive glass content. Cytocompatibility study (cell proliferation, cell attachment, alkaline phosphatase activity and osteocalcin production) using human MG-63 osteoblast-like cells in osteogenic and non-osteogenic medium showed that the composite substrates are suitable for cell attachment, proliferation and differentiation.