Distinct Cellular Mechanisms Underlie Smooth Muscle Turnover in Vascular Development and Repair.

RATIONALE: Vascular smooth muscle turnover has important implications for blood vessel repair and for the development of cardiovascular diseases, yet lack of specific transgenic animal models has prevented it's in vivo analysis. OBJECTIVE: The objective of this study was to characterize the dynamics and mechanisms of vascular smooth muscle turnover from the earliest stages of embryonic development to arterial repair in the adult. METHODS AND RESULTS: We show that CD146 is transiently expressed in vascular smooth muscle development. By using CRISPR-Cas9 genome editing and in vitro smooth muscle differentiation assay, we demonstrate that CD146 regulates the balance between proliferation and differentiation. We developed a triple-transgenic mouse model to map the fate of NG2+CD146+ immature smooth muscle cells. A series of pulse-chase experiments revealed that the origin of aortic vascular smooth muscle cells can be traced back to progenitor cells that reside in the wall of the dorsal aorta of the embryo at E10.5. A distinct population of CD146+ smooth muscle progenitor cells emerges during embryonic development and is maintained postnatally at arterial branch sites. To characterize the contribution of different cell types to arterial repair, we used 2 injury models. In limited wire-induced injury response, existing smooth muscle cells are the primary contributors to neointima formation. In contrast, microanastomosis leads to early smooth muscle death and subsequent colonization of the vascular wall by proliferative adventitial cells that contribute to the repair. CONCLUSIONS: Extensive proliferation of immature smooth muscle cells in the primitive embryonic dorsal aorta establishes the long-lived lineages of smooth muscle cells that make up the wall of the adult aorta. A discrete population of smooth muscle cells forms in the embryo and is postnatally sustained at arterial branch sites. In response to arterial injuries, existing smooth muscle cells give rise to neointima, but on extensive damage, they are replaced by adventitial cells.

Strategies to Reduce Ischemia Reperfusion Injury in Vascularized Composite Allotransplantation of the Limb.

An important and often underinvestigated contributor to solid organ transplant rejection is ischemia reperfusion injury. This pathophysiological response releases damaging reactive oxygen species and cell stress signals that initiate inflammation, which has a critical role in priming the immune system for allorecognition. In time, this renders graft dysfunction and how this response is mediated in composite tissues remains unknown. Current protocols are drawn from solid organ transplantation with little scientific basis as to how this informs current hand transplantation practices. In addition to preservation flush and allograft cooling, machine perfusion is placing itself experimentally as a concept that could act to promote viability and increase the critical ischemic window, which is especially beneficial at a time of limited donors. With the increasing prevalence worldwide of hand transplantation, we review the potential contribution of ischemia reperfusion injury to hand allograft rejection including both current and experimental strategies.

Engineering a cell-hydrogel-fibre composite to mimic the structure and function of the tendon synovial sheath.

The repair of tendon injuries is often compromised by post-operative peritendinous adhesions. Placing a physical barrier at the interface between the tendon and the surrounding tissue could potentially solve this problem by reducing adhesion formation. At present, no such system is available for routine use in clinical practice. Here, we propose the development of a bilayer membrane combining a nanofibrous poly(ε-caprolactone) (PCL) electrospun mesh with a layer of self-assembling peptide hydrogel (SAPH) laden with type-B synoviocytes. This bilayer membrane would act as an anti-adhesion system capable of restoring tendon lubrication, while assisting with synovial sheath regeneration. The PCL mesh showed adequate mechanical properties (Young's modulus=19±4 MPa, ultimate tensile stress=9.6±1.7 MPa, failure load=0.5±0.1 N), indicating that the membrane is easy to handle and capable to withstand the frictional forces generated on the tendon's surface during movement (~0.3 N). Morphological analysis confirmed the generation of a mesh with nanosized PCL fibres and small pores (< 3 µm), which prevented fibroblast infiltration to impede extrinsic healing but still allowing diffusion of nutrients and waste. Rheological tests showed that incorporation of SAPH layer allows good lubrication properties when the membrane is articulated against porcine tendon or hypodermis, suggesting that restoration of tendon gliding is possible upon implantation. Moreover, viability and metabolic activity tests indicated that the SAPH was conducive to rabbit synoviocyte growth and proliferation over 28 days of 3D culture, sustaining cell production of specific matrix components, particularly hyaluronic acid. Synoviocyte-laden peptide hydrogel promoted a sustained endogenous production of hyaluronic acid, providing an anti-friction layer that potentially restores the tendon gliding environment.

A Quantitative Systematic Review of Clinical Outcome Measure Use in Peripheral Nerve Injury of the Upper Limb.

BACKGROUND: Peripheral nerve injury (PNI) is common, leading to reduced function, pain, and psychological impact. Treatment has not progressed partly due to inability to compare outcomes between centers managing PNI. Numerous outcome measures exist but there is no consensus on which outcome measures to use nor when. OBJECTIVE: To perform a systematic review in order to describe and classify outcome measures used in PNI. METHODS: A search of Ovid Medline, Ovid Embase, Allied and Complementary Medicine Database (AMED), and CENTRAL (Cochrane Clinical Trials) was conducted. Randomized control trials (RCTs), cohort studies, and case-controlled and case series (≥5 participants) published from inception of the database until 2019 investigating adult patients with a traumatic upper limb PNI in which an outcome measurement was utilized were included. RESULTS: A total of 96 studies were included (15 RCTs, 8 case-control studies, 18 cohort studies, 5 observational studies, and the remainder were case series or retrospective reviews). A total of 56 individual outcome measures were identified, utilized across 28 different countries and 7097 patients. Ten core domains were defined: sensory subjective, sensory objective, motor subjective, motor objective, sensorimotor function, psychology and well-being, disability, quality of life, pain and discomfort, and neurotrophic measures. CONCLUSION: Lack of consensus on outcome measure use hinders comparison of outcomes between nerve injury centers and the development of novel treatments. Development of a core outcome set will help standardize outcome reporting, improve translation of novel treatments from lab to clinical practice, and ensure future research in PNI is more amenable to systematic review and meta-analysis.

The cellular biology of flexor tendon adhesion formation: an old problem in a new paradigm.

Intrasynovial flexor tendon injuries of the hand can frequently be complicated by tendon adhesions to the surrounding sheath, limiting finger function. We have developed a new tendon injury model in the mouse to investigate the three-dimensional cellular biology of intrasynovial flexor tendon healing and adhesion formation. We investigated the cell biology using markers for inflammation, proliferation, collagen synthesis, apoptosis, and vascularization/myofibroblasts. Quantitative immunohistochemical image analysis and three-dimensional reconstruction with cell mapping was performed on labeled serial sections. Flexor tendon adhesions were also assessed 21 days after wounding using transmission electron microscopy to examine the cell phenotypes in the wound. When the tendon has been immobilized, the mouse can form tendon adhesions in the flexor tendon sheath. The cell biology of tendon healing follows the classic wound healing response of inflammation, proliferation, synthesis, and apoptosis, but the greater activity occurs in the surrounding tissue. Cells that have multiple "fibripositors" and cells with cytoplasmic protrusions that contain multiple large and small diameter fibrils can be found in the wound during collagen synthesis. In conclusion, adhesion formation occurs due to scarring between two damaged surfaces. The mouse model for flexor tendon injury represents a new platform to study adhesion formation that is genetically tractable.

Aberrant Differentiation of Human Pluripotent Stem Cell-Derived Kidney Precursor Cells inside Mouse Vascularized Bioreactors.

BACKGROUND: Numerous studies have documented the in vitro differentiation of human pluripotent stem cells (hPSCs) into kidney cells. Fewer studies have followed the fates of such kidney precursor cells (KPCs) inside animals, a more life-like setting. Here, we tested the hypothesis that implanting hPSC-derived KPCs into an in vivo milieu surgically engineered to be highly vascular would enhance their maturation into kidney tissues. METHODS: 3D printed chambers containing KPCs were implanted into the thighs of adult immunodeficient mice. In some chambers, an arterial and venous flow-through (AVFT) was surgically fashioned. After 3 weeks and 3 months, implants were studied by histology, using qualitative and quantitative methods. RESULTS: After 3 weeks, chambers containing AVFTs were richer in small vessels than contralateral chambers without AVFTs. Glomeruli with capillary loops and diverse types of tubules were detected in all chambers. At 3 months, chambers contained only rudimentary tubules and glomeruli that appeared avascular. In chambers with AVFTs, prominent areas of muscle-like cells were also detected near tubules and the abnormal tissues immunostained for transforming growth factor ß1. These features have similarities to renal dysplasia, a typical histological signature of human congenital kidney malformations. CONCLUSIONS: This study urges a note of caution regarding the in vivo fates of hPSC-derived kidney precursors, with pathological differentiation appearing to follow a period of increased vascularity.

Current Limitations of Surgical Robotics in Reconstructive Plastic Microsurgery.

Surgical robots have the potential to provide surgeons with increased capabilities, such as removing physiologic tremor, scaling motion and increasing manual dexterity. Several surgical specialties have subsequently integrated robotic surgery into common clinical practice. Plastic and reconstructive microsurgical procedures have not yet  benefitted significantly from technical developments observed over the last two decades. Several studies have successfully demonstrated the feasibility of utilising surgical robots in plastic surgery procedures, yet limited work has been done to identify and analyse current barriers that have prevented wide-scale adaptation of surgical robots for microsurgery. Therefore, a systematic review using PubMed, MEDLINE, Embase and Web of Science databases was performed, in order to evaluate current state of surgical robotics within the field of reconstructive microsurgery and their limitations. Despite the theoretical potential of surgical robots, current commercially available robotic systems are suboptimal for plastic or reconstructive microsurgery. Absence of bespoke microsurgical instruments, increases in operating time, and high costs associated with robotic-assisted provide a barrier to using such systems effectively for reconstructive microsurgery. Consequently, surgical robots provide currently little overall advantage over conventional microsurgery. Nevertheless, if current barriers can be addressed and systems are specifically designed for microsurgery, surgical robots may have the potential of meaningful impact on clinical outcomes within  this surgical subspeciality.

Four-Dimensional Imaging of Soft Tissue and Implanted Biomaterial Mechanics: A Barbed Suture Case Study for Tendon Repair.

Timely, recent developments in X-ray microcomputed tomography (XµCT) imaging such as increased resolution and improved sample preparation enable nondestructive time-lapse imaging of polymeric biomaterials when implanted in soft tissue, which we demonstrate herein. Imaging the full three-dimensional (3D) structure of an implanted biomaterial provides new opportunities to assess the micromechanics of the interface between the implant and tissues and how this changes over time as force is applied in load-bearing musculoskeletal applications. In this paper, we present a case study demonstrating in situ XµCT and finite element analysis, using a dynamically loaded barbed suture repair for its novel use in tendon tissue. The aim of this study was to identify the distribution of stress in the suture and tendon as load is applied. The data gained demonstrate a clear 3D visualization of microscale features in both the tissue and implant in wet conditions. XµCT imaging has revealed, for the first time, pores around the suture, preventing full engagement of all the barbs with the tendon tissue. Subsequent finite element analysis reveals the localized stress and strain, which are not evenly distributed along the suture, or throughout the tissue. This case study demonstrates for the first time a powerful in situ mechanical imaging tool, which could be readily adapted by other laboratories to interrogate and optimize the interface between the implanted biomaterials and the soft tissue.

Versatility and "flap efficiency" of pedicled perforator flaps in lower extremity reconstruction.

BACKGROUND: The use of pedicled perforator flaps provides an alternative to free tissue transfer for lower limb reconstruction. We use computer-aided image analysis to investigate the versatility of pedicled perforator flaps for the reconstruction of lower limb defects. PATIENTS AND METHODS: Between April 2007 and April 2011, a case series of 61 patients with wounds of the lower extremity from knee to ankle were reconstructed with pedicled perforator flaps. We performed 16 pedicled reverse-flow anterolateral thigh (RF-ALT) flaps, 8 pedicled medial sural artery perforator (MSAP) flaps, 26 pedicled peroneal artery perforator (PAP) flaps, and 11 pedicled posterior tibial artery perforator (PTAP) flaps. Digital planimetry of defects covered was analyzed and the "efficiency" of each flap was calculated, which allowed the assessment of the merits of each flap in the management of lower limb defects. RESULTS: Flaps healed primarily in 82% of cases (50/61). Approximately 50% of the secondary donor sites required skin grafting. Complications requiring secondary surgery occurred in 18% (11/61) of the cases. Six required secondary skin grafting (10%). One RF-ALT flap was converted into a free flap, one PAP required arterial supercharging, and three pedicled RF-ALT flaps required venous supercharging. Image analysis showed that these pedicled perforator flaps could cover 75% of the surface area of the lower leg. The higher length of perforator allowed for greater "flap efficiency" and better versatility of tissue cover. CONCLUSION: Image analysis can be used as a modality to assess the versatility of individual flaps in the reconstruction of lower limb defects.

Improving Outcomes in Tendon Repair: A Critical Look at the Evidence for Flexor Tendon Repair and Rehabilitation.

LEARNING OBJECTIVES: After reading this article, the participant should be able to: 1. Appreciate the variation and evolution of flexor tendon management 2. Know how to assess the patient who presents with a flexor tendon laceration. 3. Understand the biology of repairing flexor tendon lacerations. 4. Appreciate the technical challenges in flexor tendon repair relating to different zones. 5. Understand the rationale of postoperative hand therapy. 6. Have an overview of the types of secondary tendon surgery. BACKGROUND: Flexor tendon injury constitutes a considerable trauma workload for hand surgeons, and a vast amount of research is dedicated toward improving outcomes in tendon repair. This Continuing Medical Education article aims to provide an up-to-date evidence-based outline of flexor tendon surgery in the hand. METHODS: The authors reviewed the literature on flexor tendon repairs to include a balanced overview of the experimental and clinical research. For each section, the best levels of evidence were assessed in the context of past research to provide a comprehensive opinion on best management. RESULTS: The review highlights current trends in flexor tendon surgery, clinical assessment, anesthetic technique, surgical approach, repair technique, and rehabilitation. Carefully selected illustrations, figures, tables, and video have been used to supplement the findings of the review. CONCLUSIONS: Early active mobilization remains the only long-term proven strategy to improve outcomes. Incorporating intraoperative mobilization using "wide-awake" surgery could emerge to further improve tendon outcomes. Good surgical approach, meticulous surgery, up-to-date physiotherapy regimens, and patient education remain the cornerstone of obtaining best outcomes.

Sutured tendon repair; a multi-scale finite element model.

Following rupture, tendons are sutured to reapproximate the severed ends and permit healing. Several repair techniques are employed clinically, with recent focus towards high-strength sutures, permitting early active mobilisation thus improving resultant joint mobility. However, the arrangement of suture repairs locally alters the loading environment experienced by the tendon. The extent of the augmented stress distribution and its effect on the tissue is unknown. Stress distribution cannot be established using traditional tensile testing, in vivo, or ex vivo study of suture repairs. We have developed a 3D finite element model of a Kessler suture repair employing multiscale modelling to represent tendon microstructure and incorporate its highly orthotropic behaviour into the tissue description. This was informed by ex vivo tensile testing of porcine flexor digitorum profundus tendon. The transverse modulus of the tendon was 0.2551 ± 0.0818 MPa and 0.1035 ± 0.0454 MPa in proximal and distal tendon samples, respectively, and the interfibrillar tissue modulus ranged from 0.1021 to 0.0416 MPa. We observed an elliptically shaped region of high stress around the suture anchor, consistent with a known region of acellularity which develop 72 h post-operatively and remain for at least a year. We also observed a stress shielded region close to the severed tendon ends, which may impair collagen fibre realignment during the remodelling stage of repair due to the lack of tensile stress.

Improving results of flexor tendon repair and rehabilitation.

BACKGROUND: The global time and effort attributed to improving outcomes in the management of flexor tendon injury are large, but the degree of advancement made over the past 50 years is relatively small. This review examines the current perceived wisdom in this field and aims to explore the limitations to the authors' understanding of the tendon healing process, examining how this may be a factor that has contributed to the authors' modest progress in the field. METHODS: The authors critically evaluate the sum of laboratory and clinical literature on the topic of zone II flexor tendon management that has guided their practice and provide evidence to support their methods. RESULTS: The review highlights some of the key developments over the years and assesses their influence on changing current practice. It also highlights recent innovations, which have the potential to influence flexor tendon outcomes by altering the surgical approach, techniques, and rehabilitation regimens. Future innovations in the field will also be discussed to examine their potential in expanding the development in the management of flexor tendon injury. CONCLUSIONS: A better understanding of flexor tendon biology will allow progress in developing new therapies for flexor tendon injuries; however, there are as yet few real breakthroughs that will dramatically change current practice.

Reduction of tendon adhesions following administration of Adaprev, a hypertonic solution of mannose-6-phosphate: mechanism of action studies.

Repaired tendons may be complicated by progressive fibrosis, causing adhesion formation or tendon softening leading to tendon rupture and subsequent reduced range of motion. There are few therapies available which improve the gliding of damaged tendons in the hand. We investigate the role of Mannose 6-phosphate (M6P) in a 600 mM hypertonic solution (Adaprev) on tendon adhesion formation in vivo using a mouse model of severed tendon in conjunction with analysis of collagen synthesis, cellular proliferation and receptors involved in TGF beta signalling. Cytotoxicity was assessed by measuring tissue residency, mechanical strength and cell viability of tendons after treatment with Adaprev. To elicit potential modes of action, in vitro and ex vivo studies were performed investigating phosphorylation of p38, cell migration and proliferation. Adaprev treatment significantly (p<0.05) reduced the development of adhesions and improved collagen organisation without reducing overall collagen synthesis following tendon injury in vivo. The bioavailability of Adaprev saw a 40% reduction at the site of administration over 45 minutes and tendon fibroblasts tolerated up to 120 minutes of exposure without significant loss of cell viability or tensile strength. These favourable effects were independent of CI-MPR and TGF-ß signalling and possibly highlight a novel mechanism of action related to cellular stress demonstrated by phosphorylation of p38. The effect of treatment reduced tendon fibroblast migration and transiently halted tendon fibroblast proliferation in vitro and ex vivo. Our studies demonstrate that the primary mode of action for Adaprev is potentially via a physical, non-chemical, hyperosmotic effect.