Modular, Vascularized Hypertrophic Cartilage Constructs for Bone Tissue Engineering Applications.

Insufficient vascularization is a main barrier to creating engineered bone grafts for treating large and ischemic defects. Modular tissue engineering approaches have promise in this application because of the ability to combine tissue types and to localize microenvironmental cues to drive desired cell function. In direct bone formation approaches, it is challenging to maintain sustained osteogenic activity, since vasculogenic cues can inhibit tissue mineralization. This study harnessed the physiological process of endochondral ossification to create multiphase tissues that allowed concomitant mineralization and vessel formation. Mesenchymal stromal cells in pellet culture were differentiated toward a cartilage phenotype, followed by induction to chondrocyte hypertrophy. Hypertrophic pellets exhibited increased alkaline phosphatase activity, calcium deposition, and osteogenic gene expression relative to chondrogenic pellets. In addition, hypertrophic pellets secreted and sequestered angiogenic factors, and supported new blood vessel formation by co-cultured endothelial cells and undifferentiated stromal cells. Multiphase constructs created by combining hypertrophic pellets and vascularizing microtissues and maintained in unsupplemented basal culture medium were shown to support robust vascularization and sustained tissue mineralization. These results demonstrate a new in vitro strategy to produce multiphase engineered constructs that concomitantly support the generation of mineralize and vascularized tissue in the absence of exogenous osteogenic or vasculogenic medium supplements.

Pre-cultured, cell-encapsulating fibrin microbeads for the vascularization of ischemic tissues.

There is a significant clinical need to develop effective vascularization strategies for tissue engineering and the treatment of ischemic pathologies. In patients afflicted with critical limb ischemia, comorbidities may limit common revascularization strategies. Cell-encapsulating modular microbeads possess a variety of advantageous properties, including the ability to support prevascularization in vitro while retaining the ability to be injected in a minimally invasive manner in vivo. Here, fibrin microbeads containing human umbilical vein endothelial cells (HUVEC) and bone marrow-derived mesenchymal stromal cells (MSC) were cultured in suspension for 3 days (D3 PC microbeads) before being implanted within intramuscular pockets in a SCID mouse model of hindlimb ischemia. By 14 days post-surgery, animals treated with D3 PC microbeads showed increased macroscopic reperfusion of ischemic foot pads and improved limb salvage compared to the cellular controls. Delivery of HUVEC and MSC via microbeads led to the formation of extensive microvascular networks throughout the implants. Engineered vessels of human origins showed evidence of inosculation with host vasculature, as indicated by erythrocytes present in hCD31+ vessels. Over time, the total number of human-derived vessels within the implant region decreased as networks remodeled and an increase in mature, pericyte-supported vascular structures was observed. Our findings highlight the potential therapeutic benefit of developing modular, prevascularized microbeads as a minimally invasive therapeutic for treating ischemic tissues.

REMAP Periop: a randomised, embedded, multifactorial adaptive platform trial protocol for perioperative medicine to determine the optimal enhanced recovery pathway components in complex abdominal surgery patients within a US healthcare system.

INTRODUCTION: Implementation of enhanced recovery pathways (ERPs) has resulted in improved patient-centred outcomes and decreased costs. However, there is a lack of high-level evidence for many ERP elements. We have designed a randomised, embedded, multifactorial, adaptive platform perioperative medicine (REMAP Periop) trial to evaluate the effectiveness of several perioperative therapies for patients undergoing complex abdominal surgery as part of an ERP. This trial will begin with two domains: postoperative nausea/vomiting (PONV) prophylaxis and regional/neuraxial analgesia. Patients enrolled in the trial will be randomised to arms within both domains, with the possibility of adding additional domains in the future. METHODS AND ANALYSIS: In the PONV domain, patients are randomised to optimal versus supraoptimal prophylactic regimens. In the regional/neuraxial domain, patients are randomised to one of five different single-injection techniques/combination of techniques. The primary study endpoint is hospital-free days at 30 days, with additional domain-specific secondary endpoints of PONV incidence and postoperative opioid consumption. The efficacy of an intervention arm within a given domain will be evaluated at regular interim analyses using Bayesian statistical analysis. At the beginning of the trial, participants will have an equal probability of being allocated to any given intervention within a domain (ie, simple 1:1 randomisation), with response adaptive randomisation guiding changes to allocation ratios after interim analyses when applicable based on prespecified statistical triggers. Triggers met at interim analysis may also result in intervention dropping. ETHICS AND DISSEMINATION: The core protocol and domain-specific appendices were approved by the University of Pittsburgh Institutional Review Board. A waiver of informed consent was obtained for this trial. Trial results will be announced to the public and healthcare providers once prespecified statistical triggers of interest are reached as described in the core protocol, and the most favourable interventions will then be implemented as a standardised institutional protocol. TRIAL REGISTRATION NUMBER: NCT04606264.

Use of Nanotechnology as an Alternative to Opioids for Post-operative Pain Management Following TKA.

Introduction: In response to the current opioid crisis, there is a great interest to develop non-pharmacologic approaches to post-operative pain management for patients undergoing total knee arthroplasty (TKA). Case Report: We report a case of a patient whose post-operative pain following TKA was managed using the newly designed NeuroCuple™ device, a patch based on the use of nanotechnology. The NeuroCuple™ device was placed above the knee by the patient at home. The use of the NeuroCuple™ device allowed the patient completely avoid the use of any opioids at home. Conclusion: Our report suggests that the use of the NeuroCuple™ device may represent an interesting alternative to opioids for the peri-operative management of pain following a primary, unilateral TKA.

Perioperative truncal peripheral nerve blocks for bariatric surgery: an opioid reduction strategy.

BACKGROUND: Bariatric surgical patients are vulnerable to cardiopulmonary depressant effects of opioids. The enhanced recovery after surgery (ERAS) protocol to improve postoperative morbidity recommends regional anesthesia for postoperative pain management. However, there is limited evidence that peripheral nerve blocks (PNB) have added benefit. OBJECTIVE: Study the effect of PNB on postoperative pain and opioid use following bariatric surgery. SETTING: Academic medical center, United States. METHODS: We conducted a cohort study of patients who underwent sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB) surgery. A total of 44 patients received the control ERAS protocol with preoperative oral extended-release morphine sulfate (MS), while 45 patients underwent a PNB with either intrathecal morphine (IM) or oral MS per local ERAS protocol. The PNB group either underwent preoperative bilateral T7 paravertebral (PVT) PNBs (27 patients) with IM or postoperative transversus abdominis plane (TAP) PNBs (18 patients) with oral MS. The primary outcome compared total opioid consumption between the ERAS control group and the PNB group up to 48 hours postoperatively. Secondary outcomes included comparison by block type and postoperative pain scores. RESULTS: PVT or TAP PNB patients had a reduction in mean postoperative oral morphine equivalent (OME) requirements compared with the ERAS protocol cohort at 24 hours (93.9 versus 42.8 mg), P < .0001; at 48 hours (72.6 versus 40.5 mg); and in pain scores at 24 hours (5.64/10 versus 4.46/10), P = .02. OME and pain scores were higher in the SG cohort. CONCLUSION: Addition of truncal PNB to standard ERAS protocol for bariatric surgical patients reduces postoperative total opioid consumption.

Multimodular vascularized bone construct comprised of vasculogenic and osteogenic microtissues.

Bioengineered bone designed to heal large defects requires concomitant development of osseous and vascular tissue to ensure engraftment and survival. Adult human mesenchymal stromal cells (MSC) are promising in this application because they have demonstrated both osteogenic and vasculogenic potential. This study employed a modular approach in which cells were encapsulated in biomaterial carriers (microtissues) designed to support tissue-specific function. Osteogenic microtissues consisting of MSC embedded in a collagen-chitosan matrix; vasculogenic (VAS) microtissues consisted of endothelial cells and MSC in a fibrin matrix. Microtissues were precultured under differentiation conditions to induce appropriate MSC lineage commitment, and were then combined in a surrounding fibrin hydrogel to create a multimodular construct. Results demonstrated the ability of microtissues to support lineage commitment, and that preculture primes the microtissues for the desired function. Combination of osteogenic and vasculogenic microtissues into multimodular constructs demonstrated that osteogenic priming resulted in sustained osteogenic activity even when cultured in vasculogenic medium, and that vasculogenic priming induced a pericyte-like phenotype that resulted in development of a primitive vessel network in the constructs. The modular approach allows microtissues to be separately precultured to harness the dual differentiation potential of MSC to support both bone and blood vessel formation in a unified construct.

Evaluation and Analysis of Fellow Learning and Education Curriculum in a Regional Anesthesiology and Acute Pain Medicine Fellowship: A Prospective, Observational Pilot Study.

Background: Since 2017, several regional anesthesiology and acute pain medicine fellowship programs throughout the country have developed various educational didactic curriculums to address the core medical knowledge requirements as set by the Accreditation Council for Graduate Medical Education. Given the paucity of existing literature regarding the medical knowledge acquisition of regional anesthesiology and acute pain medicine fellows, this study aimed to determine how quickly these fellows learn during their fellowship year, with a secondary aim of analyzing a new educational didactic curriculum in its goal of delivering the required medical knowledge. Methods: An 89-question, multiple-choice examination was administered to the 2020-2021 regional anesthesiology and acute pain medicine fellows at the University of Pittsburgh Medical Center during orientation and again at 4 months and 8 months into the fellowship. A secondary analysis of anonymous deidentified answers was completed. Results: Fellows averaged 64%, 74%, and 79% correct responses on the orientation, 4-month, and 8-month exams, respectively. An analysis of the orientation exam revealed that the most commonly incorrect answers stemmed from topics including lower extremity nerve blocks, truncal blocks, and neuraxial anesthesia. The 4-month exam showed overall marked improvement; however, truncal blocks remained the most missed topic. Topics with 100% correct response rates in all examinations were local anesthetic pharmacology and systemic opioids. Conclusions: The results of this study indicate that a large portion of learning occurs during the first 4 months of the fellowship and slows thereafter. Using this simple form of fellowship evaluation, changes to an educational didactic curriculum can be implemented to reach medical knowledge goals more effectively and efficiently as required by the Accreditation Council for Graduate Medical Education.

Coculture of Endothelial and Stromal Cells to Promote Concurrent Osteogenesis and Vasculogenesis.

A key challenge in the treatment of large bone defects is the need to provide an adequate and stable vascular supply as new tissue develops. Bone tissue engineering applies selected biomaterials and cell types to create an environment that promotes tissue formation, maturation, and remodeling. Mesenchymal stromal cells (MSCs) have been widely used in these strategies because of their established effects on bone formation, and their ability to act as stabilizing pericytes that support vascular regeneration by endothelial cells (ECs). However, the creation of vascularized bone tissue in vitro requires coupling of osteogenesis and vasculogenesis in a three-dimensional (3D) biomaterial environment. In the present study, 3D fibrin hydrogels containing MSCs and ECs were prevascularized in vitro for 7 days to create an endothelial network in the matrix, and were subsequently cultured for a further 14 days under either continued vasculogenic stimulus, a combination of vasculogenic and osteogenic (hybrid) stimulus, or only osteogenic stimulus. It was found that ECs produced robust vessel networks in 3D fibrin matrices over 7 days of culture, and these networks continued to expand over the 14-day treatment period under vasculogenic conditions. Culture in hybrid medium resulted in maintenance of vessel networks for 14 days, while osteogenic culture abrogated vessel formation. These trends were mirrored in data representing overall cell viability and cell number in the 3D fibrin constructs. MSCs were found to colocalize with EC networks under vasculogenic and hybrid conditions, suggesting pericyte-like function. The bone marker alkaline phosphatase increased over time in hybrid and osteogenic media, but mineral deposition was evident only under purely osteogenic conditions. These results suggest that hybrid media compositions can support some aspects of multiphase tissue formation, but that alternative strategies are needed to obtain robust, concomitant vascularization, and osteogenesis in engineered tissues in vitro. Impact statement The combined use of mesenchymal stromal cells (MSCs) and endothelial cells to concomitantly produce mature bone and a nourishing vasculature is a promising tissue engineering approach to treating large bone defects. However, it is challenging to create and maintain vascular networks in the presence of osteogenic cues. This study used a 3D fibrin matrix to demonstrate that prevascularization of the construct can lead to maintenance of vessel structures over time, but that osteogenesis is compromised under these conditions. This work illuminates the capacity of MSCs to serve as both supportive pericytes and as osteoprogenitor cells, and motivates new strategies for coupling osteogenesis and vasculogenesis in engineered bone tissues.

Coupling Osteogenesis and Vasculogenesis in Engineered Orthopedic Tissues.

Inadequate vascularization of engineered tissue constructs is a main challenge in developing a clinically impactful therapy for large, complex, and recalcitrant bone defects. It is well established that bone and blood vessels form concomitantly during development, as well as during repair after injury. Endothelial cells (ECs) and mesenchymal stromal cells (MSCs) are known to be key players in orthopedic tissue regeneration and vascularization, and these cell types have been used widely in tissue engineering strategies to create vascularized bone. Coculture studies have demonstrated that there is crosstalk between ECs and MSCs that can lead to synergistic effects on tissue regeneration. At the same time, the complexity in fabricating, culturing, and characterizing engineered tissue constructs containing multiple cell types presents a challenge in creating multifunctional tissues. In particular, the timing, spatial distribution, and cell phenotypes that are most conducive to promoting concurrent bone and vessel formation are not well understood. This review describes the processes of bone and vascular development, and how these have been harnessed in tissue engineering strategies to create vascularized bone. There is an emphasis on interactions between ECs and MSCs, and the culture systems that can be used to understand and control these interactions within a single engineered construct. Developmental engineering strategies to mimic endochondral ossification are discussed as a means of generating vascularized orthopedic tissues. The field of tissue engineering has made impressive progress in creating tissue replacements. However, the development of larger, more complex, and multifunctional engineered orthopedic tissues will require a better understanding of how osteogenesis and vasculogenesis are coupled in tissue regeneration. Impact statement Vascularization of large engineered tissue volumes remains a challenge in developing new and more biologically functional bone grafts. A better understanding of how blood vessels develop during bone formation and regeneration is needed. This knowledge can then be applied to develop new strategies for promoting both osteogenesis and vasculogenesis during the creation of engineered orthopedic tissues. This article summarizes the processes of bone and blood vessel development, with a focus on how endothelial cells and mesenchymal stromal cells interact to form vascularized bone both during development and growth, as well as tissue healing. It is meant as a resource for tissue engineers who are interested in creating vascularized tissue, and in particular to those developing cell-based therapies for large, complex, and recalcitrant bone defects.

Preoperative Pelvic Floor Injections With Bupivacaine and Dexamethasone for Pain Control After Vaginal Prolapse Repair: A Randomized Controlled Trial.

OBJECTIVE: To test the hypothesis that preoperative pelvic floor muscle injections and pudendal nerve blocks with bupivacaine and dexamethasone would decrease postoperative pain after vaginal native tissue prolapse repairs, compared with saline and bupivacaine. METHODS: We conducted a three-arm, double-blind, randomized trial of bilateral transobturator levator ani muscle injections and transvaginal pudendal nerve blocks before vaginal reconstructive and obliterative prolapse procedures (uterosacral ligament suspension, sacrospinous ligament fixation, levator myorrhaphy, or colpocleisis). Women were randomized to one of three study medication groups: 0.9% saline, 0.25% bupivacaine, or combination 0.25% bupivacaine with 4 mg dexamethasone. Our primary outcome was a numeric rating scale pain score on postoperative day 1. Using an analysis of variance evaluated at the two-sided 0.05 significance level, an assumed variance of the means of 0.67, and SD of 1.75, we calculated 21 women per arm to detect a 2-point change on the numeric rating scale (90% power), which we increased to 25 per arm to account for 20% attrition and the use of nonparametric statistical methods. RESULTS: From June 2017 through April 2019, 281 women were screened and 75 (26.7%) were randomized with no differences in baseline demographics among study arms. There was no significant difference in median pain scores on postoperative day 1 among study groups (median [interquartile range] pain score 4.0 [2.0-7.0] for placebo vs 4.0 [2.0-5.5] for bupivacaine vs 4.0 [1.5-5.0] for bupivacaine with dexamethasone, P=.92). CONCLUSION: Preoperative pelvic floor muscle injections and pudendal nerve blocks with bupivacaine and dexamethasone did not improve postoperative pain after vaginal native tissue prolapse procedures. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT03040011.

Injectable osteogenic microtissues containing mesenchymal stromal cells conformally fill and repair critical-size defects.

Repair of complex fractures with bone loss requires a potent, space-filling intervention to promote regeneration of bone. We present a biomaterials-based strategy combining mesenchymal stromal cells (MSC) with a chitosan-collagen matrix to form modular microtissues designed for delivery through a needle to conformally fill cavital defects. Implantation of microtissues into a calvarial defect in the mouse showed that osteogenically pre-differentiated MSC resulted in complete bridging of the cavity, while undifferentiated MSC produced mineralized tissue only in apposition to native bone. Decreasing the implant volume reduced bone regeneration, while increasing the MSC concentration also attenuated bone formation, suggesting that the cell-matrix ratio is important in achieving a robust response. Conformal filling of the defect with microtissues in a carrier gel resulted in complete healing. Taken together, these results show that modular microtissues can be used to augment the differentiated function of MSC and provide an extracellular environment that potentiates bone repair.

Thromboelastography in term neonates.

BACKGROUND: Thromboelastography (TEG) is utilized as a point-of-care test of coagulation function to improve evidence-based blood product replacement in adults. In contrast to standard indices of coagulation, TEG reflects the dynamic interactions among the elements involved in hemostasis, including fibrinolysis. Although normal adult values and various abnormalities have been characterized, normative values for term neonates have not been described. Studies of neonatal TEG remain limited and have small sample sizes with inconsistent methodology. The aim of this study is to provide normative data on healthy term neonates, and to assess the impact of mode of delivery on TEG parameters at term. METHODS: Venous umbilical blood was obtained from the placenta within 10 min of delivery. TEG analysis of citrated kaolin-activated samples was performed for 50 healthy term vaginal and 50 cesarean deliveries. Samples collected for cesarean sections were from scheduled cases or unscheduled ones due to failure of progression of labor. RESULTS: Healthy neonates with uncomplicated vaginal term deliveries resulted in the following TEG parameters: R: 5.41 ± 1.34 (mean ± SD) min; K: 1.62 ± 0.75 min; α-angle: 65.39 ± 8.77°; MA: 65.86 ± 5.81 mm; and LY30: 1.40 ± 1.18%. Results for the cesarean delivery neonatal TEG assays showed: R: 5.51 ± 1.74 (mean ± SD) min; K: 1.52 ± 0.47 min; α-angle: 64.15 ± 4.61°; MA: 64.15 ± 4.61 mm; and LY30: 2.44 ± 3.51%. Of note, no statistical differences were observed (p < .01) between the groups. CONCLUSION: TEG measurements from term neonates were no different when the neonates were delivered vaginally or by cesarean section. Labor had no effect on neonatal TEG levels. Neonatal TEG values may therefore serve as insight for fetal values at the appropriate postconceptional age.

Neurotoxicity of common peripheral nerve block adjuvants.

PURPOSE OF REVIEW: This review outlines the analgesic role of perineural adjuvants for local anesthetic nerve block injections, and evaluates current knowledge regarding whether adjuvants modulate the neurocytologic properties of local anesthetics. RECENT FINDINGS: Perineural adjuvant medications such as dexmedetomidine, clonidine, buprenorphine, dexamethasone, and midazolam play unique analgesic roles. The dosing of these medications to prevent neurotoxicity is characterized in various cellular and in-vivo models. Much of this mitigation may be via reducing the dose of local anesthetic used while achieving equal or superior analgesia. Dose-concentration animal models have shown no evidence of deleterious effects. Clinical observations regarding blocks with combined bupivacaine-clonidine-buprenorphine-dexamethasone have shown beneficial effects on block duration and rebound pain without long-term evidence of neurotoxicity. In-vitro and in-vivo studies of perineural clonidine and dexmedetomidine show attenuation of perineural inflammatory responses generated by local anesthetics. SUMMARY: Dexmedetomidine added as a peripheral nerve blockade adjuvant improves block duration without neurotoxic properties. The combined adjuvants clonidine, buprenorphine, and dexamethasone do not appear to alter local anesthetic neurotoxicity. Midazolam significantly increases local anesthetic neurotoxicity in vitro, but when combined with clonidine-buprenorphine-dexamethasone (sans local anesthetic) produces no in-vitro or in-vivo neurotoxicity. Further larger-species animal testing and human trials will be required to reinforce the clinical applicability of these findings.

Failure of Intraoperative Red Cell Salvage: A Patient with Sickle Cell Disease and HELLP (Hemolysis, Elevated Liver enzymes and Low Platelets) Syndrome.

Cell salvage is a process whereby the bloodshed from the operative field is collected and returned to the patient. It can be especially useful when allogeneic red blood cell (RBC) units are not readily available such as when the recipient has multiple alloantibodies. We report on the anesthesia and transfusion strategies for managing a pregnant patient with sickle cell disease (SCD) with HELLP (Hemolysis, Elevated Liver enzymes and Low Platelets) syndrome. A pregnant patient with twins at 30 weeks of gestation was admitted in an SCD crisis. She subsequently developed HELLP syndrome and required urgent cesarean delivery; however, she had multiple RBC antibodies complicating the immediate provision of cross-matched RBC units. Cell salvage was used to capture the blood shed during her procedure while the blood bank was searching for compatible RBCs units. Despite multiple interventions designed to optimize the cell salvage procedure for the unique challenges of a patient with SCD, the salvaged RBCs hemolyzed and could not be reinfused. Cell salvage in an obstetric patient with SCD in an acute crisis and super-imposed HELLP was unable to recover intact and useable RBCs. Further studies into methods of optimizing the procedure for use in this context are warranted. Close communication between the clinical teams treating the patient and the transfusion service is required so that the RBC transfusion requirements can be anticipated; this is especially important when the patient has multiple antibodies.

The cost of resident scholarly activity and its effect on resident clinical experience.

BACKGROUND: Scholarly activity is an important aspect of the academic training of future anesthesiologists. However, residents' scholarly activity may reduce training caseloads and increase departmental costs. METHODS: We conducted this study within a large academic anesthesiology residency program with data from the 4 graduating classes of 2009 through 2012. Scholarly activity included peer-reviewed manuscripts, case reports, poster presentations at conferences, book chapters, or any other publications. It was not distinguished whether a resident was the principal investigator or a coinvestigator on a project. The following data were collected on each resident: months spent on a resident research rotation, number of scholarly projects completed, number of research conferences attended, and Accreditation Council for Graduate Medical Education case entries. Comparison was made between residents electing a resident research rotation with those who did not for (1) scholarly projects, (2) research conference attendance, and (3) Accreditation Council for Graduate Medical Education case numbers. Cost to the department for extra clinical coverage during residents' time spent on research activities was calculated using an estimated average cost of $675 ± $176 (mean ± SD) per day with local certified registered nurse anesthetist pay scales. RESULTS: Sixty-eight residents were included in the analyses. Twenty-four residents (35.3%) completed resident research rotations with an average duration of 3.7 months. Residents who elected resident research rotations completed more scholarly projects (5 projects [4-6]: median [25%-75% interquartile range] vs 2 [0-3]; P < 0.0001), attended more research conferences (2 conferences [2-4] vs 1 [0-2]; P < 0.0001), but experienced fewer cases (980 cases [886-1333] vs 1182 [930-1420]; P ≤ 0.002) compared with those who did not elect resident research rotations. The estimated average cost to the department per resident who elected a resident research rotation was $13,500 ± $9724 per month. The average resident time length away from duty for conference attendance was 3.2 ± 0.2 days, with an average cost to the department of $2160 ± $565. The average annual departmental expense for resident conference travel was an additional $1424 ± $133 per resident, as calculated from reimbursement data. Together, the estimated departmental cost for resident scholarly activity during the residency training period was $27,467 ± $20,153 per resident. CONCLUSIONS: Residents' scholarly activities require significant departmental financial support. Residents who elected to spend months conducting research completed significantly more scholarly projects but experienced fewer clinical cases.