Role of Adult Tissue-Derived Pluripotent Stem Cells in Bone Regeneration.

BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNC) consist of a heterogeneous mix of mesenchymal stem cells (MSC), hematopoietic progenitor cells (HPC), endothelial progenitor cells (EPC), monocytes, lymphocytes and pluripotent stem cells. Whereas the importance of MSC and EPC has been well documented in bone healing and regeneration studies, the role of pluripotent stem cells is still poorly understood. In the present study we evaluated if and how Very Small Embryonic Like cells (VSEL), isolated from rat BM-MNC, contribute to bone healing. METHODS: Large bone defects were made in the femurs of 38 Sprague Dawley female rats and treated with ß-TCP scaffold granules seeded with male VSEL; BM-MNC, VSEL-depleted BM-MNC or scaffold alone, and bone healing was evaluated at 8 weeks post-surgery. RESULTS: Bone healing was significantly increased in defects treated with VSEL and BM-MNC, compared to defects treated with VSEL-depleted BM-MNC. Donor cells were detected in new bone tissue, in all the defects treated with cells, and in fibrous tissue only in defects treated with VSEL-depleted BM-MNC. The number of CD68+ cells was the highest in the VSEL-depleted group, whereas the number of TRAP positive cells was the lowest in this group. CONCLUSIONS: Based on the results, we can conclude that VSEL play a role in BM-MNC induced bone formation. In our rat femur defect model, in defects treated with VSEL-depleted BM-MNC, osteoclastogenesis and bone formation were decreased, and foreign body reaction was increased.

Electrical stimulation shifts healing/scarring towards regeneration in a rat limb amputation model.

Different species respond differently to severe injury, such as limb loss. In species that regenerate, limb loss is met with complete restoration of the limbs' form and function, whereas in mammals the amputated limb's stump heals and scars. In in vitro studies, electrical stimulation (EStim) has been shown to promote cell migration, and osteo- and chondrogenesis. In in vivo studies, after limb amputation, EStim causes significant new bone, cartilage and vessel growth. Here, in a rat model, the stumps of amputated rat limbs were exposed to EStim, and we measured extracellular matrix (ECM) deposition, macrophage distribution, cell proliferation and gene expression changes at early (3 and 7 days) and later stages (28 days). We found that EStim caused differences in ECM deposition, with less condensed collagen fibrils, and modified macrophage response by changing M1 to M2 macrophage ratio. The number of proliferating cells was increased in EStim treated stumps 7 days after amputation, and transcriptome data strongly supported our histological findings, with activated gene pathways known to play key roles in embryonic development and regeneration. In conclusion, our findings support the hypothesis that EStim shifts injury response from healing/scarring towards regeneration. A better understanding of if and how EStim controls these changes, could lead to strategies that replace scarring with regeneration.

Necrotizing fasciitis: treatment concepts and clinical results.

BACKGROUND: Necrotizing fasciitis is a life-threatening soft tissue infection characterized by a rapid spreading infection of the subcutaneous tissue and in particular the fascia. The management of infected tissues requires a rapid diagnosis, immediate aggressive surgical management and an extended debridement. In some cases early amputations of the affected tissues and maximum intensive care treatment, in case of sepsis, are required. Due to a rising number of cases we aimed to evaluate our patients in a retrospective review. METHOD: All patients diagnosed with necrotizing fasciitis from 2014 to 2016 (21 months) in our level one trauma center were identified. Their charts were reviewed and data were analyzed in terms of demographic and social information, microbiological results, therapeutic course, socio-economic outcome and mortality. RESULTS: We found 15 patients with necrotizing fasciitis. None of these died in the observation period. The mean number of surgical interventions was seven. Two patients underwent limb amputation; diabetes mellitus was assigned with a significant higher risk for amputation. The mean hospitalization was 32 days, including 8 days on intensive care unit. Of the discovered bacteria 93% were sensitive to the initial antibiotic treatment with Ampicillin, Clindamycin and Clont. CONCLUSION: Surgical therapy is indicated if necrotizing fasciitis is suspected. Diabetes mellitus was a clinical predictor of limb amputation in patients with necrotizing fasciitis in our cohort. Aminopenicillin ± sulbactam in combination with clindamycin and/or metronidazole is recommended as initial calculated antibiotic treatment.

Frankfurt microsurgery course: the first 175 trainees.

INTRODUCTION: Microsurgery courses, taught external to surgical training programs, are essential for acquiring the high level of technical skill required for clinical proficiency. METHODS: The Frankfurt microsurgery course is a 5-day, intensive course that teaches arterial and venous anastomosis using end-to-end, end-to-side, one-way-up, continuous-suture, and vessel graft techniques. During the course, the instructor records the level of skill (in-course data) achieved by each trainee by assessing anastomosis completion and patency. Demographic information is also collected. Post-course trainees are invited to complete an online survey (post-course data) to get their opinions of the courses' effectiveness. RESULTS: The in-course "skill achievement" and post-course "course effectiveness" data are presented below. In-course data: 94.8 and 59.9% of participants completed patent end-to-end arterial and venous anastomoses, respectively, while 85.4% performed a patent end-to-side anastomosis. 96.1 and 57.1% of participants who attempted arterial and venous anastomoses using the one-way-up technique were successful, as were 90.9% of those attempting continuous-suture technique. Patent venous grafts were performed by 54.7% of participants. POST-COURSE DATA: All respondents indicated significant improvement of their microsurgical skills after taking the course. 66.7% of respondents considered the full-time presence of the instructor to be the most valuable aspect of the course. All respondents would highly recommend the course to colleagues. CONCLUSION: The microcourse significantly increased trainees' clinical microsurgery skills, confidence, and the number of clinical cases they perform. Of all the anastomosis techniques taught, venous anastomosis and grafting were the most difficult to learn. The presence of a full-time experienced instructor was most important.

A review of thrombosis and antithrombotic therapy in microvascular surgery.

Despite a remarkable expansion of microsurgery, there is still no international consensus about routinely used prophylactic antithrombotic agents. Most treatment regimens still use aspirin, heparin (low-molecular-weight and unfractionated heparin) or colloids (hydroxyphenylacetate 6%/dextran); however, clear evidence for the clinical benefit of an ideal administration regimen or one agent over the other has not yet been established. Instead of searching for the one regime that fits all, an increasing number of reviews from different disciplines describe multistep approaches that optimize what has been shown to be most promising. This includes the use of antithrombotic agents, proper risk assessment, secondary prevention and professional training to optimize microsurgical skills. In this review, we describe factors included in traditional approaches and also emphasize the value of good surgical technique, which while recognized by all to be one of the most important factors for success, receives less emphasis in reviews describing thrombosis prophylaxis in microvascular surgery.

Endothelial progenitor cells improve directly and indirectly early vascularization of mesenchymal stem cell-driven bone regeneration in a critical bone defect in rats.

Early vascularization of a composite in a critical bone defect is a prerequisite for ingrowth of osteogenic reparative cells to regenerate bone, since lack of vessels does not ensure a sufficient nutritional support of the bone graft. The innovation of this study was to investigate the direct and indirect effects of endothelial progenitor cells (EPCs) and cotransplanted mesenchymal stem cells (MSCs) on the in vivo neovascularization activity in a critical size defect at the early phase of endochondral ossification. Cultivated human EPCs and MSCs were loaded onto ß-TCP in vitro. A critical-sized bone defect (5 mm) was created surgically in the femoral diaphysis of adult athymic rat and stabilized with an external fixateur. The bone defects were filled with ß-TCP, MSCs seeded on ß-TCP, EPCs seeded on ß-TCP, and coculture of MSCs and EPCs seeded on ß-TCP or autologous bone of rat. After 1 week, the rats were sacrificed. Using quantitative CD34 immunohistochemistry as well as qualitative analysis of vascularization (staining of MHC and VEGF) in decalcified serial sections were performed by means of an image analysis system. Fluorescence microscopy analyzed the direct effects and indirect effects of human implanted EPCs for vessel formation at bone regeneration site. Formation of a primitive vascular plexus was also detectable in the ß-TCP, MSC, or autologous bone group, but on a significantly higher level if EPCs alone or combined with MSCs were transplanted. Moreover, highest amount of vascularization were detected when EPCs and MSCs together were implanted. Early vascularization is improved by transplanted EPCs, which formed new vessels directly. Indeed the indirect effect of EPCs to vascularization is much higher. Transplanted EPC release chemotactic factors (VEGF) to recruit EPCs of the host and stimulate vascularization in the bone defect. Transplantation of human EPCs displays a promising approach to improve early vascularization of a scaffold in a critical bone defect. Moreover, coculture of EPCs and MSCs demonstrate also a synergistic effect on new vessel formation and seems to be a potential osteogenic construct for in vivo application.

Risk assessment and management in hand and facial tissue transplantation.

PURPOSE: Since hand and facial tissue transplantation are new treatments, risk data must be derived from early reports of the few cases done to date combined with extrapolations from other procedures with similar risks. This manuscript summarizes data from both sources including eight separate studies that measure the real and perceived risks associated with hand and facial tissue transplantation. METHODS: Real Risks: Several large clinical studies describing risk data from kidney transplant recipients (10-years experience) and risk data from 49 human hand transplants in 33 recipients (>10 years experience) were reviewed. Perceived Risks: Over 500 subjects with different life experiences (facially disfigured, amputees, laryngectomees, kidney transplant recipients, transplant and reconstructive surgeons, and controls) were surveyed using a standardized and validated risk assessment instrument. RESULTS: Real Risks: Contrary to earlier estimates the risks of acute rejection are higher (80-85%) while the risks of chronic rejection (<5%) and other complications are lower than previously reported. Perceived Risks: Despite different life experiences, those questioned would accept similar amounts of risk to receive different types of transplant procedures (hand, foot, face, larynx, and kidney) but all would accept the most risk for a facial tissue transplant. Kidney transplant recipients, who live with the risks of immunosuppression, would accept the most risk while facially disfigured individuals and reconstructive surgeons, who have real-life experience with facial disfigurement, would accept the least. CONCLUSIONS: These studies contribute to the growing body of risk data necessary for moving hand and face transplantation into mainstream medicine.

Mesenchymal stem cell (MSC) and endothelial progenitor cell (EPC) growth and adhesion in six different bone graft substitutes.

INTRODUCTION: Several different synthetic and allograft bone graft substitutes are used clinically to treat large bone defects. In contrast to the "gold standard" of autologous bone grafts, these do not contain bone-forming (MSC) or vessel-forming (EPC) cells. In order to achieve the same level of success enjoyed by autologous bone grafts, they must be compatible with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC). In a previous study, we seeded MSC onto six different bone graft substitutes and then measured the cell adhesion, viability, differentiation, and morphology. In the present study, we seeded both MSC and EPC onto the same six bone graft substitutes and measured the same parameters. METHODS: In vitro, 125,000 MSC and 125,000 EPC were seeded onto Chronos(®), Vitoss(®), Actifuse(®), Biobase(®), Cerabone(®), and Tutoplast(®). Cell adhesion (fluorescence microscopy) and viability (MTT assay) were measured on days 2, 6, and 10. Osteogenic (cbfa-1, alkaline phosphatase [ALP], osteocalcin, collagen-1 alpha [Col1A]) and endothelial (von Willebrand factor [vWF], vascular endothelial growth factor [VEGF], kinase domain receptor [KDR]) gene expression were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Morphology was described by scanning electron microscopy (SEM) at day 2. RESULTS: MSC adhered significantly better to Tutoplast(®), Chronos(®), Actifuse(®), and Biobase(®). EPC adhered better to Actifuse(®), Chronos(®), Biobase(®), and Tutoplast(®). Viability increased over time when seeded on Tutoplast(®) and Chronos(®). Osteogenic and endothelial gene expression were detectable at day 10 in cells seeded on Chronos(®), Actifuse(®), and Tutoplast(®). The best morphology of MSC and EPC was found on Tutoplast(®), Chronos(®), Actifuse(®), and Biobase(®). CONCLUSION: When bone graft substitutes are used to help fill large defects, it is important that their interaction with these cells be supportive of bone healing.

Population structure of the beetle pests Phyllodecta vulgatissima and P. vitellinae on UK willow plantations.

Phyllodecta (= Phratora) vulgatissima and P. vitellinae (Coleoptera: Chrysomelidae) are important pests of willows and poplars. Their differences in host species preference may provide a non-chemical control strategy for pest control. However, little is known about population structure with respect to hosts, regions or seasons. Using five microsatellites, 850 P. vulgatissima and 1100 P. vitellinae individuals, comprising 17 and 22 UK samples, respectively, were genotyped. High diversity was observed at all loci. Migrant numbers exchanged per generation (Nm) were high (2.1-12.6 for P. vulgatissima and 0.9-12.2 for P. vitellinae), suggesting high genetic exchange between samples. Estimates of population differentiation (FST) and analyses of the data using Bayesian methods (Partition and Structure) showed little evidence of subdivision in relation to geography, sampling time or host.

Co-segregation analysis of cadmium and zinc accumulation in Thlaspi caerulescens interecotypic crosses.

• Cadmium (Cd) hyperaccumulation in Thlaspi caerulescens varies among ecotypes. Here we investigated segregation of Cd and zinc (Zn) accumulation in F2 crosses between high (Ganges) and low (Prayon) Cd-accumulating ecotypes. • Accumulation was measured in plants grown in compost treated with 5 and 100 mg kg-1 Cd and Zn, respectively, and in hydroponics with 50 m Zn and 10 or 50 m Cd. Another hydroponic experiment examined the relationship between Cd tolerance and accumulation. • Parental phenotype distributions for shoot metal concentrations were distinct for Cd, but not consistent for Zn. Shoot Cd and Zn in F2 s varied continuously, with significant transgression for Zn in all treatments. Shoot Cd correlated strongly with shoot manganese (Mn), and to a lesser degree with shoot Zn. Shoot Cd concentrations in the Cd nontolerant F2 s were lower than, or similar to, those in the Cd-tolerant F2 s. • We conclude that Cd and Zn accumulation is governed by multiple genes, and that Cd tolerance and accumulation are independent traits in T. caerulescens. Two uptake systems with distinctive affinities for Cd, Zn and Mn are proposed.

The role of phytochelatins in arsenic tolerance in the hyperaccumulator Pteris vittata.

• Pteris vittata was the first identified arsenic (As) hyperaccumulator. Here we investigated whether phytochelatins (PCs) are involved in the hypertolerance of arsenic by P. vittata. • P. vittata was exposed to 0-500 µm arsenate for 5 d, or to 50 µm arsenate for 0-7 d. In addition, l-buthionine-sulphoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase, was used in combination with different arsenate exposures. The relationships between As accumulation and the concentrations of PCs and glutathione (GSH) were examined. • PC synthesis was induced upon exposure to arsenate in P. vittata, with only PC2 detected in the plant. The As concentration correlated significantly with PC2 concentration in both roots and shoots, but not with GSH. The molar ratio of PC-SH to As was c. 0.09 and 0.03 for shoots and roots, respectively, suggesting that only a small proportion (1-3%) of the As in P. vittata can be complexed with PCs. In the presence of arsenate, addition of BSO decreased PC2 concentrations in roots and shoots by 89-96% and 30-33%, respectively. BSO alone was found to inhibit root growth of P. vittata markedly. • The results suggest that PCs play a limited role in the hypertolerance of As in P. vittata.

Dynamic rectus abdominis muscle flap for intestinal stomal continence: a systematic approach.

Several attempts to create a continent stomal sphincter using dynamic myoplasty with limited success have been reported. Denervation atrophy and early muscle fatigue have plagued all reported attempts to make a continent stoma a reality. To address this problem in a series of experiments, we designed a stomal sphincter using the most caudal segment of the rectus abdominis muscle. Then, we performed a study to determine whether a sphincter created with a rectus abdominis muscle island flap could maintain stomal continence in the short term. We found that when stimulated using two different electrical stimulation protocols, in all cases the rectus abdominis muscle sphincter generated peak pressures well above those needed to maintain stomal continence (60 mm Hg). All sphincters were able to maintain stomal continence at all intraluminal bowel pressures tested. We found one of these protocols to be far superior and reached 4 hours of stomal continence after 8 to 10 weeks of electrical stimulation.

Multi-channel orbicularis oculi stimulation to restore eye-blink function in facial paralysis.

Facial paralysis due to facial nerve injury results in the loss of function of the muscles of the hemiface. The most serious complication in extreme cases is the loss of vision. In this study, we compared the effectiveness of single- and multiple-channel electrical stimulation to restore a complete and cosmetically acceptable eye blink. We established bilateral orbicularis oculi muscle (OOM) paralysis in eight dogs; the OOM of one side was directly stimulated using single-channel electrical stimulation and the opposite side was stimulated using multi-channel electrical stimulation. The changes in the palpebral fissure and complete palpebral closure were measured. The difference in current intensities between the multi-channel and single-channel simulation groups was significant, while only multi-channel stimulation produced complete eyelid closure. The latest electronic stimulation circuitry with high-quality implantable electrodes will make it possible to regulate precisely OOM contractions and thus generate complete and cosmetically acceptable eye-blink motion in patients with facial paralysis.

Gracilis muscle neosphincter for treating urinary incontinence.

The purpose of this study was to test the anatomical and functional feasibility of using a gracilis muscle free flap to create a urinary sphincter. Anatomical studies were performed in 12 human cadavers and short-term (n = 7) and long-term (n = 8) functional studies were performed in dogs. In the short-term functional studies, the left gracilis muscle was transferred into the pelvis and wrapped around the urethra and the right gracilis muscle was wrapped around a stent. A cuff electrode was placed on the muscle's nerve pedicle and used to stimulate the neosphincter while peak pressure, fatigue rate, and perfusion measurements were performed. In the long-term functional studies, intramuscular electrodes were inserted into the neosphincter to stimulate the flap. The flaps were wrapped around the urethra and dogs were followed for 16 weeks, during which time urodynamic measurements were performed. Our anatomical studies demonstrated that the gracilis muscle free flap could be transferred into the pelvis to create a urinary neosphincter. Our short-term functional study demonstrated that gracilis muscle free-flap function and perfusion were not compromised by transfer. In our long-term functional study, all neosphincters provided bladder outlet resistance pressures consistent with continence. Our anatomical, short-term, and long-term functional studies indicate that a gracilis muscle free-flap neosphincter is an effective procedure for treating urinary incontinence.

Microsatellite analysis of the inbreeding grass weed Barren Brome (Anisantha sterilis) reveals genetic diversity at the within- and between-farm scales.

Nine microsatellites were used to screen 131 samples of Barren Brome (Anisantha sterilis: synonym Bromus sterilis) collected from within the fields of three English farms [from Oxfordshire (Oxon), Leicestershire (Leics) and Wiltshire (Wilts)] and eight seeds taken from samples of each of 10 farms across England, UK. Most individuals (approximately 97%) were homozygous. Polymorphism occurred at all nine loci in all three farms sampled at the field scale, and at most loci for nine of the other 10 farm samples. Between three and 11 alleles were found per locus. Gene diversity (D = 1 - summation operator p(i)2) ranged from 0.088 to 0.760. Polymorphism occurred among individuals within and among fields, and farms. Some alleles were found in only one farm. On the basis of the alleles at all nine loci in the 211 sampled plants, a total of 92 (44%) different genotypes was identified. Clustering analysis using the unweighted pair group method with arithmetic averages (UPGMA) for the combined Oxon, Wilts and Leics samples did not cluster them into their respective farms. Similarly, a phenogram of samples from all 10 farms showed considerable mixing of individuals with respect to farm origins. Identification of genotypes on field plans showed evidence of both spatial localization and mixing. Previous reports have suggested that A. sterilis is strictly inbreeding with little intrapopulation variation at the genetic level. Our data reveal that A. sterilis exists as numerous separate and genetically different lines, which are maintained by inbreeding but which very occasionally outcross. Possible explanations for this pattern of high genetic diversity are discussed.

Respect for persons, informed consent and the assessment of infectious disease risks in xenotransplantation.

Given the increasing need for solid organ and tissue transplants and the decreasing supply of suitable allographic organs and tissue to meet this need, it is understandable that the hope for successful xenotransplantation has resurfaced in recent years. The biomedical obstacles to xenotransplantation encountered in previous attempts could be mitigated or overcome by developments in immunosuppression and especially by genetic manipulation of organ source animals. In this essay we consider the history of xenotransplantation, discuss the biomedical obstacles to success, explore recent developments in transgenic sourcing of organs and tissues, and analyze the problem of infectious disease resulting from xenotransplantation (xenosis). We then apply a model of risk analysis to these risks. The conclusions of this risk analysis are used in an ethical evaluation of informed consent in xenotransplantation, with an ethical foundation in Kantian autonomy and Levinasian heteronomic alterity. Our conclusion is that individual and collective informed consent to the infectious disease risks of xenotransplantation requires an open, participatory and dialogical public policy process not yet seen in the United States and Europe. Until that process is created, we propose caution in xenotransplantation in general and a postponement of solid organ xenotransplants in particular.

Dynamic rectus abdominis muscle sphincter for stoma continence: an acute functional study in a dog model.

Fecal stomal incontinence is a problem that continues to defy surgical treatment. Previous attempts to create continent stomas using dynamic myoplasty have had limited success due to denervation atrophy of the muscle flap used in the creation of the sphincter and because of muscle fatigue resulting from continuous electrical stimulation. To address the problem of denervation atrophy, a stomal sphincter was designed using the most caudal segment of the rectus abdominis muscle, preserving its intercostal innervation as well as its vascular supply. The purpose of the present study was to determine whether this rectus abdominis muscle island flap sphincter design could maintain stomal continence acutely. In this experiment, six dogs were used to create eight rectus abdominis island flap stoma sphincters around a segment of distal ileum. Initially, the intraluminal stomal pressures generated by the sphincter using different stimulation frequencies were determined. The ability of this stomal sphincter to generate continence at different intraluminal bowel pressures was then assessed. In all cases, the rectus abdominis muscle sphincter generated peak pressures well above those needed to maintain stomal continence (60 mmHg). In addition, each sphincter was able to maintain stomal continence at all intraluminal bowel pressures tested.

Construction of a linkage map of the Rennell Island Tall coconut type (Cocos nucifera L.) and QTL analysis for yield characters.

AFLP and SSR DNA markers were used to construct a linkage map in the coconut (Cocos nucifera L.; 2n = 32) type Rennell Island Tall (RIT). A total of 227 markers were arranged into 16 linkage groups. The total genome length corresponded to 1971 cM for the RIT map, with 5-23 markers per linkage group. QTL analysis for yield characters in two consecutive sampling periods identified nine loci. Three and two QTLs were detected for number of bunches and one and three QTLs for number of nuts. The correlation of trait values between characters and evaluation periods is partially reflected in identical QTLs. The QTLs represent characters that are important in coconut breeding. The cosegregation of markers with these QTLs provides an opportunity for marker-assisted selection in coconut breeding programmes.