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.

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.

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.