Reduction of Activin A gives rise to comparable expression of key definitive endoderm and mature beta cell markers.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

M inverted V glansplasty: an update on technique and outcomes 30 years later.

Distal hypospadias is a common congenital urology anomaly for which numerous corrective procedures have been described. Over the last 40 years, the gold-standard operative technique for distal hypospadias has switched from the meatal advancement and glanuloplasty (MAGPI) procedure to the tubularized incised plate (TIP) urethroplasty. A modification to the MAGPI procedure, first described 30 years ago, is the M inverted V (MIV) glansplasty, which improved upon the MAGPI procedure to reduce instances of meatal retraction. The MIV glansplasty is unique compared to many commonly used procedures as it does not require a formal urethroplasty or incorporation of a dartos flap, and it does not always necessitate as extensive mobilization of the glans wings. We describe our updated technique and outcomes of the MIV glansplasty and delineate situations where the MIV is best employed.

PEGDA microencapsulated allogeneic islets reverse canine diabetes without immunosuppression.

BACKGROUND: Protection of islets without systemic immunosuppression has been a long-sought goal in the islet transplant field. We conducted a pilot biocompatibility/safety study in healthy dogs followed by a dose-finding efficacy study in diabetic dogs using polyethylene glycol diacrylate (PEGDA) microencapsulated allogeneic canine islets. METHODS: Prior to the transplants, characterization of the canine islets included the calculations determining the average cell number/islet equivalent. Following measurements of purity, insulin secretion, and insulin, DNA and ATP content, the islets were encapsulated and transplanted interperitoneally into dogs via a catheter, which predominantly attached to the omentum. In the healthy dogs, half of the microspheres injected contained canine islets, the other half of the omentum received empty PEGDA microspheres. RESULTS: In the biocompatibility study, healthy dogs received increasing doses of cells up to 1.7 M cells/kg body weight, yet no hypoglycemic events were recorded and the dogs presented with no adverse events. At necropsy the microspheres were identified and described as clear with attachment to the omentum. Several of the blood chemistry values that were abnormal prior to the transplants normalized after the transplant. The same observation was made for the diabetic dogs that received higher doses of canine islets. In all diabetic dogs, the insulin required to attempt to control blood glucose was cut by 50-100% after the transplant, down to no required insulin for the course of the 60-day study. The dogs had no adverse events and behavioral monitoring suggested normal activity after recovery from the transplant. CONCLUSIONS AND IMPLICATIONS: The study provides evidence that PEGDA microencapsulated canine islets reversed the signs of diabetes without immunosuppression and led to states of insulin-independence or significantly lowered insulin requirements in the recipients.

Association of Operating Room Costs With Head and Neck Surgical Instrumentation Optimization: A Surgeon-Led Quality Improvement Initiative.

Importance: In an era of increasing health care expenditure, reduction of redundant head and neck surgical instrumentation may minimize waste. Objective: To (1) optimize head and neck surgical instrument trays at a single large academic tertiary care center and (2) identify associated direct cost savings. Design, Setting, and Participants: This prospective quality improvement initiative was conducted at an academic tertiary care medical center from July 2017 through July 2021. Participants were a multidisciplinary surgical quality team consisting of head and neck surgeons, operating room nurses, surgical technicians, and supply chain analysts. Main Outcomes and Measures: The 4 primary surgical instrument trays (major otolaryngology [Oto], Oto plastics, direct laryngoscopy, and microdirect laryngoscopy) used in all head and neck procedures were reviewed by 10 head and neck surgical faculty with detailed case observation of instrument utilization performed by trained operating room nurses and surgical technologists. Instruments used in less than 40% of cases were excluded from surgical trays, and novel instrument trays were established based on faculty feedback and utilization. Data on instrument processing, utilization, and associated institutional direct costs were prospectively collected over a 3-year period. The primary outcome measure was change in operating room direct costs. Surgeon satisfaction with the quality improvement intervention was the secondary outcome. Direct cost savings were identified as a function of surgical volume, labor and supply costs, and instrument depreciation. Results: More than 1500 eligible surgical cases were reviewed during the preintervention period. Of the 149 instruments in the major Oto tray, only 118 (79%) instruments were used in more than 40% of cases. There were 58 (49%) and 32 (21%) instruments in this tray that were used in more than 40% of neck dissections and sentinel lymph node biopsies, respectively. Resulting intervention included development of a streamlined major Oto tray with 118 instruments and novel neck dissection and sentinel lymph node biopsy trays. Similar processes were applied to the remaining head and neck trays, with a total of 257 instruments removed. Over a 3-year postintervention period, streamlined surgical trays were used 9284 times with direct cost savings of $228 338 (95% CI, $227 817-$228 854). Overall surgeon satisfaction with the optimized head and neck surgical trays was 100%. Conclusions and Relevance: In this quality improvement study, surgeon-led elimination of redundant or rarely used instruments from surgical instrument trays was associated with reduced operating room direct costs while maintaining stakeholder satisfaction.

Hyaluronic Acid Hydrogel Microspheres for Slow Release Stem Cell Delivery.

Cell therapies are hampered by a lack of available delivery systems, resulting in inconsistent outcomes in animal studies and human clinical trials. Hydrogel encapsulants offer a broad range of tunable characteristics in the design of cell delivery vehicles. The focus of the hydrogel field has been on durable encapsulants that provide long-term paracrine function of the cells. However, some cell therapies require cell-to-cell contact in order to elicit their effect. Controlled release microencapsulants would be beneficial in these situations, but appropriate polymers have not been adaptable to microsphere manufacturing because they harden too slowly. We developed and tested a novel microencapsulant formulation (acrylated hyaluronic acid: AHA) with degradation characteristics as a controlled release cell delivery vehicle. The properties of AHA microspheres were evaluated and compared to those of poly(ethylene glycol) diacrylate (PEGDA), a durable hydrogel. AHA microspheres possessed a higher swelling ratio, lower diffusion barrier, faster degradation rate, a lower storage modulus, and a larger average diameter than microspheres composed of PEGDA. Additionally, in vitro cell viability and release and short-term in vivo biocompatibility in immune competent Sprague-Dawley rats was assessed for each microsphere type. Compared to PEGDA, microspheres composed of AHA resulted in significantly less foreign body response in vivo as measured by a lack of cellularity or fibrotic ring in the surrounding tissue and no cellular infiltration into the microsphere. This study illustrates the potential of AHA microspheres as a degradable cell delivery system with superior encapsulated cell viability and biocompatibility with the surrounding tissue.

A Versatile Microencapsulation Platform for Hyaluronic Acid and Polyethylene Glycol.

Cell microencapsulation is a rapidly expanding field with broad potential for stem cell therapies and tissue engineering research. Traditional alginate microspheres suffer from poor biocompatibility, and microencapsulation of more advanced hydrogels is challenging due to their slower gelation rates. We have developed a novel, noncytotoxic, nonemulsion-based method to produce hydrogel microspheres compatible with a wide variety of materials, called core-shell spherification (CSS). Fabrication of microspheres by CSS derived from two slow-hardening hydrogels, hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), was characterized. HA microspheres were manufactured with two different crosslinking methods: thiolation and methacrylation. Microspheres of methacrylated HA (MeHA) had the greatest swelling ratio, the largest average diameter, and the lowest diffusion barrier. In contrast, PEGDA microspheres had the smallest diameters, the lowest swelling ratio, and the highest diffusion barrier, while microspheres of thiolated HA had characteristics that were in between the other two groups. To test the ability of the hydrogels to protect cells, while promoting function, diabetic NOD mice received intraperitoneal injections of PEGDA or MeHA microencapsulated canine islets. PEGDA microspheres reversed diabetes for the length of the study (up to 16 weeks). In contrast, islets encapsulated in MeHA microspheres at the same dose restored normoglycemia, but only transiently (3-4 weeks). Nonencapsulated canine islet transplanted at the same dose did not restore normoglycemia for any length of time. In conclusion, CSS provides a nontoxic microencapsulation procedure compatible with various hydrogel types.

Ingrafts in hypospadias surgery: Longer-term outcomes.

BACKGROUND: The technique of hypospadias repair with a dorsal inlay graft (ingraft) was initially reserved for boys with an unfavorable glans configuration or in previously failed repairs. Although the ingraft technique has been used for some time, there is scarce literature describing its longer term outcomes. Additionally, there is minimal data comparing inner preputial skin and buccal mucosal graft outcomes in primary and reoperative surgery. OBJECTIVE: To describe longer-term outcomes in the use of buccal mucosa and inner preputial skin ingrafts for primary and revisional hypospadias repairs. STUDY DESIGN: We conducted a single-center retrospective review of our hypospadias repairs employing ingrafts along with a review of the literature. RESULTS: A total of 47 patients met inclusion criteria. Primary repair was performed in 38 patients, all with unfavorable glans characteristics at a mean age of 16 months and redo repairs in 9 boys at a mean age of 110 months. We used a buccal mucosal graft (BMG) in 19 boys and the remaining 28 were grafted with inner preputial skin. The complication rate was 32% in primary repairs and amongst those, 6 of 28 patients (21%) with skin grafts and 6 of 10 patients (60%) with BMGs suffered a complication. Only BMGs were used in reoperative situations and complications occurred in 2 of 9 cases (22%) of those cases. Overall, 8 patients (42%) with BMG and 6 patients (21%) with preputial skin ingrafts experienced a complication, at an average time of 17 months (range: 0.4-66 months) and 24 months (range: 1.1-113 months), respectively. Surgical correction of the complications resulted in resolution of symptoms in a majority of patients. DISCUSSION: In our experience, BMGs used as salvage therapy in revisional hypospadias surgery, had lower observed complication rates when compared to its use in primary repair. Inner preputial skin ingrafts for primary repair yielded an acceptable complication rate. Our study describes some of the longest follow-up times in the literature with complications observed even up to 10 years postoperatively. This reinforces the need for active long-term follow-up in reporting outcomes in hypospadias surgery.

The Flaws and Future of Islet Volume Measurements.

When working with isolated islet preparations, measuring the volume of tissue is not a trivial matter. Islets come in a large range of sizes and are often contaminated with exocrine tissue. Many factors complicate the procedure, and yet knowledge of the islet volume is essential for predicting the success of an islet transplant or comparing experimental groups in the laboratory. In 1990, Ricordi presented the islet equivalency (IEQ), defined as one IEQ equaling a single spherical islet of 150 µm in diameter. The method for estimating IEQ was developed by visualizing islets in a microscope, estimating their diameter in 50 µm categories and calculating a total volume for the preparation. Shortly after its introduction, the IEQ was adopted as the standard method for islet volume measurements. It has helped to advance research in the field by providing a useful tool improving the reproducibility of islet research and eventually the success of clinical islet transplants. However, the accuracy of the IEQ method has been questioned for years and many alternatives have been proposed, but none have been able to replace the widespread use of the IEQ. This article reviews the history of the IEQ, and discusses the benefits and failings of the measurement. A thorough evaluation of alternatives for estimating islet volume is provided along with the steps needed to uniformly move to an improved method of islet volume estimation. The lessons learned from islet researchers may serve as a guide for other fields of regenerative medicine as cell clusters become a more attractive therapeutic option.

Improved yield of canine islet isolation from deceased donors.

BACKGROUND: Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. RESULTS: Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). CONCLUSIONS: In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats.

Preservation of pancreatic islets for long-term storage of islets used for transplantation or research has long been a goal. Unfortunately, few studies on long-term islet cryopreservation (1 month and longer) have reported positive outcomes in terms of islet yield, survival and function. In general, single cells have been shown to tolerate the cryopreservation procedure better than tissues/multicellular structures like islets. Thus, we optimized a method to cryopreserve single islet cells and, after thawing, reaggregated them into islet spheroids. Cryopreserved (CP) single human islet cells formed spheroids efficiently within 3-5 days after thawing. Approximately 79% of islet cells were recovered following the single-cell cryopreservation protocol. Viability after long-term cryopreservation (4 weeks or more) was significantly higher in the CP islet cell spheroids (97.4 ± 0.4%) compared to CP native islets (14.6 ± 0.4%). Moreover, CP islet cell spheroids had excellent viability even after weeks in culture (88.5 ± 1.6%). Metabolic activity was 4-5 times higher in CP islet cell spheroids than CP native islets at 24 and 48 h after thawing. Diabetic rats transplanted with CP islet cell spheroids were normoglycemic for 10 months, identical to diabetic rats transplanted with fresh islets. However, the animals receiving fresh islets required a higher volume of transplanted tissue to achieve normoglycemia compared to those transplanted with CP islet cell spheroids. By cryopreserving single cells instead of intact islets, we achieved highly viable and functional islets after thawing that required lower tissue volumes to reverse diabetes in rats.

Hyaluronic Acid/Collagen Hydrogel as an Alternative to Alginate for Long-Term Immunoprotected Islet Transplantation.

Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were normoglycemic, but by 11 days returned to hyperglycemia. Immunohistological examination of the grafts and surrounding tissue indicated strong graft rejection. By comparison, when using the same outbred strain of rats, allogeneic transplantation of islets within the HA-COL gel reversed long-term diabetes and prevented graft rejection in all animals. Animals were sacrificed at 40, 52, 64, and 80 weeks for evaluation, and all were non-diabetic at sacrifice. Explanted grafts revealed viable islets in the transplant site as well as intact hydrogel, with little or no evidence of fibrotic overgrowth or cellular rejection. The results of these studies demonstrate great potential for HA-COL hydrogel as an alternative to sodium alginate for long-term immunoprotected islet transplantation.