Vascular Tissue Engineering Using Scaffold-Free Prevascular Endothelial-Fibroblast Constructs.

Vascularization remains a substantial limitation to the viability of engineered tissue. By comparing in vivo vascularization dynamics of a self-assembled prevascular endothelial-fibroblast model to avascular grafts, we explore the vascularization rate limitations in implants at early time intervals, during which tissue hypoxia begins to affect cell viability. Scaffold-free prevascular endothelial-fibroblast constructs (SPECs) may serve as a modular and reshapable vascular bed in replacement tissues. SPECs, fibroblast-only spheroids (FOS), and silicone implants were implanted in 54 Sprague Dawley rats and harvested at 6, 12, and 24 h (n = 5 per time point and implant type). We hypothesized that the primary endothelial networks of the SPECs allow earlier anastomosis and increased vessel formation in the interior of the implant compared to FOS and silicone implants within a 24 h window. All constructs were encapsulated by an endothelial lining at 6 h postimplantation and SPEC internal cords inosculated with the host vascular network by this time point. SPECs had a significantly higher microvascular area fraction and branch/junction density of penetrating cords at 6-12 h compared with other constructs. In addition, SPECs demonstrated perivascular cell recruitment, lumen formation, and network remodeling consistent with vessel maturation at 12-24 h; however, these implants were poorly perfused within our observation window, suggesting poor lumen patency. FOS vascular characteristics (microvessel area and penetrating cord density) increased within the 12-24 h period to represent those of the SPEC implants, suggesting a 12 h latency in host response to avascular grafts compared to prevascular grafts. Knowledge of this temporal advantage in in vitro prevascular network self-assembly as well as an understanding of the current limitations of SPEC engraftment builds on our theoretical temporal model of tissue graft vascularization and suggests a crucial time window, during which technological improvements and vascular therapy can improve engineered tissue survival.

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology.

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 µm in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2.

Connexin-based therapeutics have shown the potential for therapeutic efficacy in improving wound healing. Our previous work demonstrated that the connexin43 (Cx43) mimetic peptide juxtamembrane 2 (JM2) reduced the acute inflammatory response to a submuscular implant model by inhibiting purinergic signaling. Given the prospective application in improving tissue-engineered construct tolerance that these results indicated, we sought to determine the mechanism of action for JM2 in the present study. Using confocal microscopy, a gap-FRAP cell communication assay, and an ethidium bromide uptake assay of hemichannel function we found that the peptide reduced cell surface Cx43 levels, Cx43 gap junction (GJ) size, GJ communication, and hemichannel activity. JM2 is based on the sequence of the Cx43 microtubule binding domain, and microtubules have a confirmed role in intracellular trafficking of Cx43 vesicles. Therefore, we tested the effect of JM2 on Cx43-microtubule interaction and microtubule polymerization. We found that JM2 enhanced Cx43-microtubule interaction and that microtubule polymerization was significantly enhanced. Taken together, these data suggest that JM2 inhibits trafficking of Cx43 to the cell surface by promoting irrelevant microtubule polymerization and thereby reduces the number of hemichannels in the plasma membrane available to participate in proinflammatory purinergic signaling. Importantly, this work indicates that JM2 may have therapeutic value in the treatment of proliferative diseases such as cancer. We conclude that the targeted action of JM2 on Cx43 channels may improve the tolerance of implanted tissue-engineered constructs against the innate inflammatory response.

Inhibition of connexin 43 hemichannel-mediated ATP release attenuates early inflammation during the foreign body response.

BACKGROUND: In the last 50 years, the use of medical implants has increased dramatically. Failure of implanted devices and biomaterials is a significant source of morbidity and increasing healthcare expenditures. An important cause of implant failure is the host inflammatory response. Recent evidence implicates extracellular ATP as an important inflammatory signaling molecule. A major pathway for release of cytoplasmic ATP into the extracellular space is through connexin hemichannels, which are the unpaired constituents of gap junction intercellular channels. Blockade of hemichannels of the connexin 43 (Cx43) isoform has been shown to reduce inflammation and improve healing. We have developed a Cx43 mimetic peptide (JM2) that targets the microtubule-binding domain of Cx43. The following report investigates the role of the Cx43 microtubule-binding domain in extracellular ATP release by Cx43 hemichannels and how this impacts early inflammatory events of the foreign body reaction. METHODS: In vitro Cx43 hemichannel-mediated ATP release by cultured human microvascular endothelial cells subjected to hypocalcemic and normocalcemic conditions was measured after application of JM2 and the known hemichannel blocker, flufenamic acid. A submuscular silicone implant model was used to investigate in vivo ATP signaling during the early foreign body response. Implants were coated with control pluronic vehicle or pluronic carrying JM2, ATP, JM2+ATP, or known hemichannel blockers and harvested at 24 h for analysis. RESULTS: JM2 significantly inhibited connexin hemichannel-mediated ATP release from cultured endothelial cells. Importantly, the early inflammatory response to submuscular silicone implants was inhibited by JM2. The reduction in inflammation by JM2 was reversed by the addition of exogenous ATP to the pluronic vehicle. CONCLUSIONS: These data indicate that ATP released through Cx43 hemichannels into the vasculature is an important signal driving the early inflammatory response to implanted devices. A vital aspect of this work is that it demonstrates that targeted molecular therapeutics, such as JM2, provide the capacity to regulate inflammation in a clinically relevant system.

Predicting academic performance in surgical training.

INTRODUCTION: During surgical residency, trainees are expected to master all the 6 competencies specified by the ACGME. Surgical training programs are also evaluated, in part, by the residency review committee based on the percentage of graduates of the program who successfully complete the qualifying examination and the certification examination of the American Board of Surgery in the first attempt. Many program directors (PDs) use the American Board of Surgery In-Training Examination (ABSITE) as an indicator of future performance on the qualifying examination. Failure to meet an individual program's standard may result in remediation or a delay in promotion to the next level of training. Remediation is expensive in terms of not only dollars but also resources, faculty time, and potential program disruptions. We embarked on an exploratory study to determine if residents who might be at risk for substandard performance on the ABSITE could be identified based on the individual resident's behavior and motivational characteristics. If such were possible, then PDs would have the opportunity to be proactive in developing a curriculum tailored to an individual resident, providing a greater opportunity for success in meeting the program's standards. METHODS: Overall, 7 surgical training programs agreed to participate in this initial study and residents were recruited to voluntarily participate. Each participant completed an online assessment that characterizes an individual's behavioral style, motivators, and Acumen Index. Residents completed the assessment using a code name assigned by each individual PD or their designee. Assessments and the residents' 2013 ABSITE scores were forwarded for analysis using only the code name, thus insuring anonymity. Residents were grouped into those who took the junior examination, senior examination, and pass/fail categories. A passing score of ≥70% correct was chosen a priori. Correlations were performed using logistic regression and data were also entered into a neural network (NN) to develop a model that would explain performance based on data obtained from the TriMetrix assessments. RESULTS: A total of 117 residents' TriMetrix and ABSITE scores were available for analysis. They were divided into 2 groups of 64 senior residents and 53 junior residents. For each group, the pass/fail criteria for the ABSITE were set at 70 and greater as passing and 69 and lower as failing. Multiple logistic regression analysis was complete for pass/fail vs the TriMetrix assessments. For the senior data group, it was found that the parameter Theoretical correlates with pass rate (p < 0.043, B = -0.513, exp(B) = 0.599), which means increasing theoretical scores yields a decreasing likelihood of passing in the examination. For the junior data, the parameter Internal Role Awareness correlated with pass/fail rate (p < 0.004, B = 0.66, exp(B) = 1.935), which means that an increasing Internal Role Awareness score increases the likelihood of a passing score. The NN was able to be trained to predict ABSITE performance with surprising accuracy for both junior and senior residents. CONCLUSION: Behavioral, motivational, and acumen characteristics can be useful to identify residents "at risk" for substandard performance on the ABSITE. Armed with this information, PDs have the opportunity to intervene proactively to offer these residents a greater chance for success. The NN was capable of developing a model that explained performance on the examination for both the junior and the senior examinations. Subsequent testing is needed to determine if the NN is a good predictive tool for performance on this examination.

Purinergic signaling in early inflammatory events of the foreign body response: modulating extracellular ATP as an enabling technology for engineered implants and tissues.

Purinergic signaling is a ubiquitous and vital aspect of mammalian biology in which purines--mainly adenosine triphosphate (ATP)--are released from cells through loss of membrane integrity (cell death), exocytosis, or transport/diffusion across membrane channels, and exert paracrine or autocrine signaling effects through three subclasses of well-characterized receptors: the P1 adenosine receptors, the P2X ionotropic nucleotide receptors, and the P2Y metabotropic receptors. ATP and its metabolites are released by damaged and stressed cells in injured tissues. The early events of wound healing, hemostasis, and inflammation are highly regulated by these signals through activation of purinergic receptors on platelets and neutrophils. Recent data have demonstrated that ATP signaling is of particular importance to targeting leukocytes to sites of injury. This is particularly relevant to the subject of implanted medical devices, engineered tissues, and grafts as all these technologies elicit a wound healing response with varying degrees of encapsulation, rejection, extrusion, or destruction of the tissue or device. Here, we review the biology of purinergic signaling and focus on ATP release and response mechanisms that pertain to the early inflammatory phase of wound healing. Finally, therapeutic options are explored, including a new class of peptidomimetic drugs based on the ATP-conductive channel connexin43.

A model system for primary abdominal closures.

The foreign body response to medical devices and materials implanted in the human body, including scarring, fibrous encapsulation, and potential rejection, is a longstanding and serious clinical issue. There are no widely acceptable or safe therapies for ameliorating the foreign body response. Clinical complications resulting from the response include disfigurement of silicone prostheses and loss of function of devices such as implanted pacemakers, stents, and shunts. Cellularized implants and stem cells placed in the body are also subject to the foreign body response with the added issue that the regenerative repair intended to be prompted by the graft may be inhibited. Beneficial modification of the body's reaction to implanted materials, medical devices, engineered constructs, or stem cells would be a fundamentally important therapeutic advance.As part of investigating the cellular response, we have developed a model which uses cells isolated from skeletal muscle biopsy, cultured, and proliferated in vitro. These satellite cells, which are mononucleated progenitor cells, reside between the plasma membrane of the muscle fiber and the basal membrane that encompasses the fiber. While usually quiescent, these cells become activated following muscle damage. Once activated, the satellite cells proliferate, migrate to injured muscle, and participate in repair by fusing with existing muscle fibers or by differentiating into new skeletal muscle fibers. Satellite cells have been shown to be heterogeneous populations of stem cells and progenitor cells. We have developed an explant method for isolating, sorting, enriching, and culturing these cells for use in skeletal muscle regenerative medicine to determine if the foreign body response can be inhibited by manipulating the cell-cell communication.

Determining personal talents and behavioral styles of applicants to surgical training: a new look at an old problem, part II.

BACKGROUND: The selection of applicants for training in any particular surgical program is an imprecise exercise. Despite the abundance of information on particular candidates, many of the fundamental qualities that are associated with success for the surgical trainee cannot be identified by review of the applicants' grades, scores, letters of recommendation, personal statement, or even from the interview process. We sought a method to determine behavior, motivation, and values possessed by applicants that coincided with traits by our current residents who had demonstrated success in our program. METHODS: The methods have been described in detail in Part I.(1) Briefly, the individual applicants' personal talent report was used to develop a rank-ordered list by the outside consultant and was compared to the traditionally developed rank list developed by the Department in the traditional fashion and the newly developed job benchmark. RESULTS: Five hundred thirty-five applications were received and interviews were offered to 112 (21%) applicants. Seventy-five on-line surveys were completed by the 77 applicants who were interviewed. The consultant was able to identify important personal talents, elements of motivation, and behavioral style that were not gleaned from the application or the interview process, some of which prompted a revision of our final ranking order.(1) This report discusses the results of the motivational analysis and of the Personal Talents Skills Inventory. Applicants with a strong motivation for the theoretical (knowledge) and social commitment (desire to help others) are important characteristics. Clear views of the external world and of self, as well as a sense of satisfaction with the applicants' vision of their future are positively associated with success in our program. CONCLUSIONS: The ability to identify unique behavioral, motivational and personal talents that applicants bring to the program that were not identifiable from the traditional application and interview process has allowed us to determine applicants who were a good match for the structure and culture of our program.

Determining personal talents and behavioral styles of applicants to surgical training: a new look at an old problem, part I.

BACKGROUND: The selection of residents for any program and their evaluation for success is an inexact science. Errors can prove costly, disruptive, and potentially damaging to training programs, and personal and professional setbacks can occur for resident applicants. A method was sought to determine the intangible characteristics of applicants to a general surgery residency program, particularly an assessment of behavior and motivation. The hypothesis was that such information could contribute to a more objective analysis of how well an applicant might fit into a program and its culture, and therefore improve the residents' chance for success and reduce the attrition rate. METHODS: Applications were screened by the Program Director and selection committee according to departmental standards. Those applicants who were offered the opportunity for interview were asked to complete an on-line survey that assessed behavioral style, intrinsic motivators, and dimensional balance. The assessment is known as the TriMetrix Personal Talent Report (TriMetrix; Target Training International, Ltd; TTI, Phoenix, AZ). An initial job benchmark was constructed from data based on surveys of current residents and faculty, and from interviews held with Subject Matter Experts (SMEs) familiar with the demands of the position and the qualities necessary for success. Resident selection was carried out as has been done historically within the program. An independent contractor, who was blinded to the ranking by the program, presented an applicant list based on the candidate's Personal Talent Reports against the job benchmark. The ranking lists were then compared. RESULTS: Of the 535 applications received, interviews were offered to 112, and 77 interviews were conducted. Seventy-five on-line TriMetrix (TTI) assessments were completed by the applicants. Rank lists developed independently by the program and by the consultant were compared, with obvious discrepancies. Overall there was little concordance between the two lists, suggesting that the TriMetrix (TTI) assessment measures something different. The job benchmark identified different behavioral styles among the most successful of the current residents, suggesting that a diversity of natural behavior does not preclude success in the program. CONCLUSIONS: Objective data regarding an individual's personal style can be used to identify applicants who match with a training program's job benchmark. Factors predictive of success specific to our program include an independent desire for knowledge, a commitment to the service of others, and a view of the world with a sense of direction and purpose. The diversity of our current residents' styles as identified by this analysis indicates that many different individuals can be successful. While the instrument can provide important information regarding elements that contribute to successful performance, it is weighted as one essential component utilized in conjunction with other tools.

Swallowing dysfunction in trauma patients with cervical spine fractures treated with halo-vest fixation.

UNLABELLED: ACKGROUND:: Cervical spine fractures are common in traumatically injured patients. The halo-vest brace is a common treatment used for these fractures. We hypothesize that the use of halo-vest fixation is associated with a high incidence of dysphagia in trauma patients. METHODS: All trauma patients at our Level I Trauma Center from August 2005 to August 2007 were analyzed retrospectively via the trauma registry (N=3,702). Included were adult patients with cervical spine fractures treated with halo-vests and evaluated formally by speech-language pathologists for dysphagia and aspiration. Patients were categorized into mild, moderate, and severe dysphagia. RESULTS: Of the 3,702 patients, 369 (10%) had cervical spine fractures from blunt trauma and 56 met inclusion criteria. Of these, 19 (34%) had no evidence of swallowing dysfunction and the remaining 37 (66%) had evidence of dysphagia. Thirteen (23%) exhibited symptoms of aspiration. There were no significant differences in age, gender, Injury Severity Score, arrival Revised Trauma Score, or arrival Glasgow Coma Scale score on presentation. Dysphagia is associated with longer intensive care unit stays (p=0.019) and trends toward a longer hospital stay (p=0.083). In trauma patients with halo-vests, increasing severity of dysphagia from mild to moderate is associated with longer ventilator days (p=0.005), intensive care unit days (p=0.001), and hospital length of stay (p=0.015). CONCLUSIONS: Patients with cervical fractures treated with halo-vest fixation have a significantly high incidence of dysphagia and aspiration. Dysphagia in trauma patients treated with halo-vests for c-spine fractures is common, associated with worse outcomes, and difficult to predict. Therefore, all of these patients should be formally evaluated for dysphagia.

Human satellite progenitor cells for use in myofascial repair: isolation and characterization.

Current use of prosthetic meshes and implants for myofascial reconstruction has been associated with infectious complications, long-term failure, and dissatisfying cosmetic results. Our laboratory has developed a small animal model for ventral hernia repair, which uses progenitor cells isolated from a skeletal muscle biopsy. In the model, progenitor cells are expanded in vitro, seeded onto a nonimmunogenic, novel aligned scaffold of bovine collagen and placed into the defect as a living adjuvant to the innate repair mechanism. The purpose of the current investigation is to examine the feasibility of translating our current model to humans. As a necessary first step we present our study on the efficacy of isolating satellite cells from 9 human donor biopsies. We were able to successfully translate our progenitor cell isolation and culture protocols to a human model with some modifications. Specifically, we have isolated human satellite muscle cells, expanded them in culture, and manipulated these cells to differentiate into myotubes in vitro. Immunohistochemical analysis allowed the characterization of distinct progenitor cell cycle stages and quantification of approximate cell number. Furthermore, isolated cells were tracked via cytoplasmic nanocrystal labeling and observed using confocal microscopy.

Novel bacterial immobilization compound effectively decreases bacterial counts in healthy volunteers.

Skin flora immobilization technology is similar in efficacy to Iodine-Povidone in healthy volunteers. We did a prospective study in a university clinic with 60 healthy volunteers. Right inguinal skin area on healthy volunteers was used to compare the antimicrobial properties of cyanoacrylate sealant (FloraSeal, Adhesion Biomedical, Wyomissing, PA) versus standard surgical preparation Povidone-iodine (Betadine, Purdue Productions, Stamford, CT). Bacterial counts were measured at different time intervals: 15 minutes, 4 hours, and 24 hours. Bacterial colony forming units were compared between Povidone-iodine and cyanoacrylate sealant. The absolute log reduction was 5.568 for Povidone-iodine (7 absolute CFU); 5.028 for cyanoacrylate (59 absolute CFU); and 5.568 for Povidone-iodine and cyanoacrylate combined (21 absolute CFU). Cyanoacrylate was able to sustain a reduction on bacterial counts at 4 hours and 24 hours of more than 99.8 per cent as compared with the control group. Cyanoacrylate microbial sealant successfully reduces bacterial counts on normal healthy skin. The results were similar to Povidone-iodine alone. We believe this technology may be an excellent means of mitigating incisional surgical site infection by reducing the risk of contamination by skin flora and warrants further testing.

Focused in vivo genetic analysis of implanted engineered myofascial constructs.

Successfully engineering functional muscle tissue either in vitro or in vivo to treat muscle defects rather than using the host muscle transfer would be revolutionary. Tissue engineering is on the cutting edge of biomedical research, bridging a gap between the clinic and the bench top. A new focus on skeletal muscle tissue engineering has led investigators to explore the application of satellite cells (autologous muscle precursor cells) as a vehicle for engineering tissues either in vitro or in vivo. However, few skeletal muscle tissue-engineering studies have reported on successful generation of living tissue substitutes for functional skeletal muscle replacement. Our model system combines a novel aligned collagen tube and autologous skeletal muscle satellite cells to create an engineered tissue repair for a surgically created ventral hernia as previously reported [SA Fann, L Terracio, W Yan, et al., A model of tissue-engineered ventral hernia repair, J Invest Surg. 2006;19(3):193-205]. Several key features we specifically observe are the significant persistence of transplanted skeletal muscle cell mass within the engineered repair, the integration of new tissue with adjacent native muscle, and the presence of significant neovascularization. In this study, we report on our experience investigating the genetic signals important to the integration of neoskeletal muscle tissue. The knowledge gained from our model system applies to the repair of severely injured extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.

The use of prognostic indicators in the development of a statistical model predictive for adrenal insufficiency in trauma patients.

We performed a retrospective chart review of trauma patients admitted to Palmetto Richland Memorial Hospital and identified 63 cases of adrenal insufficiency along with 65 trauma patient controls. Two statistical models, a neural network and a multiple logistic regression, were developed to predict patients with increased risk of developing adrenal insufficiency. Each model had 11 selected independent variables, along with patient demographic data, to make a probabilistic prediction of patient outcome. The neural network model was trained with 102 patients to identify linear and nonlinear relationships that could yield a predictive capability. The neural network achieved an accuracy of 71 per cent. The logistic regression model achieved an accuracy of 82 per cent. With these models, we have shown the feasibility of a method to more accurately screen patients with an increased risk of adrenal insufficiency. This ability should allow earlier identification and treatment of patients with adrenal insufficiency. Further development with a larger database is needed to improve the accuracy of the present models.

Nanomechanical characterization of micro/nanofiber reinforced type I collagens.

To function properly in the rigorous tissue environment, implanted scaffolds for tissue engineering are required to meet certain standards of strength and mechanical integrity. However, the soft nature and moisture condition of biomaterials impose great challenges to many existing techniques and instrumentations for measuring their mechanical properties at micro/nano scale. In this work, we demonstrate the testing methodologies of micro/nano fiber reinforced type I collagens, and obtain basic mechanical property data of two types of modified collagens-micro carbon fiber reinforced collagen (MCFR) and nano collagen fiber reinforced collagen (NCFR). Results show that mechanical properties of collagen tissues can be enhanced by reinforcing nano collagen fibers but weakened by micro carbon fiber reinforcements. Mechanisms are discussed on how natures of reinforcing fibers affect reinforcement to the collagen matrix, as well as how reinforcements behave within the collagen matrix in response to mechanical strain.

A model of tissue-engineered ventral hernia repair.

We have developed a tissue-engineered ventral hernia repair system using our novel aligned collagen tube and autologous skeletal muscle satellite cells. In this model system, skeletal muscle satellite cells were isolated from a biopsy, expanded in culture, and incorporated into our collagen tube scaffold, forming the tissue-engineered construct. We characterized the results of the repaired hernias on both the gross and microscopic scales and compared them to an unrepaired control, an autologous muscle repair control, and a collagen-tube-only repair. Untreated animals developed a classic hernia sac, devoid of abdominal muscle and covered only with a thin layer of mesothelial tissue. Significant muscle, small-diameter blood vessels, and connective tissue were apparent in both the autologous control and the engineered muscle repairs. The engineered muscle repairs became cellularized, vascularized, and integrated with the native tissue, hence becoming a "living" repair. A tissue-engineered construct repair of ventral hernias with subsequent incorporation and vascularization could provide the ultimate in anterior wall myofascial defect repair and would further the understanding of striated muscle engineering. The knowledge gained from our model system would have immediate application to mangled extremities, maxillofacial reconstructions, and restorative procedures following tumor excision in other areas of the body.