In vivo characterization of 3D-printed polycaprolactone-hydroxyapatite scaffolds with Voronoi design to advance the concept of scaffold-guided bone regeneration

Three-dimensional (3D)-printed medical-grade polycaprolactone (mPCL) composite scaffolds have been the first to enable the concept of scaffold-guided bone regeneration (SGBR) from bench to bedside. However, advances in 3D printing technologies now promise next-generation scaffolds such as those with Voronoi tessellation. We hypothesized that the combination of a Voronoi design, applied for the first time to 3D-printed mPCL and ceramic fillers (here hydroxyapatite, HA), would allow slow degradation and high osteogenicity needed to regenerate bone tissue and enhance regenerative properties when mixed with xenograft material. We tested this hypothesis in vitro and in vivo using 3D-printed composite mPCL-HA scaffolds (wt 96%:4%) with the Voronoi design using an ISO 13485 certified additive manufacturing platform. The resulting scaffold porosity was 73% and minimal in vitro degradation (mass loss <1%) was observed over the period of 6 months. After loading the scaffolds with different types of fresh sheep xenograft and ectopic implantation in rats for 8 weeks, highly vascularized tissue without extensive fibrous encapsulation was found in all mPCL-HA Voronoi scaffolds and endochondral bone formation was observed, with no adverse host-tissue reactions. This study supports the use of mPCL-HA Voronoi scaffolds for further testing in future large preclinical animal studies prior to clinical trials to ultimately successfully advance the SGBR concept

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Bone responses to tissue engineered constructs (TECs) in critical large bone defects: Towards improved histological and immunohistochemical assessment

This research was focused on the development of advanced quantitative methods to evaluate the use of a 3D printed membrane scaffold, for the guidance and spatiotemporal delivery of recombinant human bone growth factor for the regeneration of a large bone defect, which still represents a major challenge in orthopaedic and reconstructive surgery. Two histomorphometric approaches were investigated and successfully applied. The combined histological, immunohistochemical and histomorphometric analyses outlined in this thesis further elucidated the mechanisms and patterns that govern the bone regeneration of a critical sized bone defect in a large experimental ovine model

Histological and Immunohistochemical Characterization of Osteoimmunological Processes in Scaffold-Guided Bone Regeneration in an Ovine Large Segmental Defect Model

Large-volume bone defect regeneration is complex and demands time to complete. Several regeneration phases with unique characteristics, including immune responses, follow, overlap, and interdepend on each other and, if successful, lead to the regeneration of the organ bone’s form and function. However, during traumatic, infectious, or neoplastic clinical cases, the intrinsic bone regeneration capacity may exceed, and surgical intervention is indicated. Scaffold-guided bone regeneration (SGBR) has recently shown efficacy in preclinical and clinical studies. To investigate different SGBR strategies over periods of up to three years, we have established a well-characterized ovine large segmental tibial bone defect model, for which we have developed and optimized immunohistochemistry (IHC) protocols. We present an overview of the immunohistochemical characterization of different experimental groups, in which all ovine segmental defects were treated with a bone grafting technique combined with an additively manufactured medical-grade polycaprolactone/tricalcium phosphate (mPCL-TCP) scaffold. The qualitative dataset was based on osteoimmunological findings gained from IHC analyses of over 350 sheep surgeries over the past two decades. Our systematic and standardized IHC protocols enabled us to gain further insight into the complex and long-drawn-out bone regeneration processes, which ultimately proved to be a critical element for successful translational research.</p

Comparison of different decalcification methods using rat mandibles as a model

Selection of decalcification agents is an essential consideration when processing mineralized tissues because the integrity and immunohistochemical characteristics of the tissues may be affected. Here, we report results obtained from the decalcification of rat mandibles using 10% ethylenediaminetetraacetic acid (EDTA) at room temperature (RT), 10% EDTA at 37C, 5% nitric acid, and 10% formic acid at RT. Decalcification endpoints were determined by microcomputed tomography. Morphological preservation and antigenicity were evaluated by hematoxylin and eosin staining and immunohistochemistry. Decalcification of the anterior and posterior portions of the mandible took 220 and 191 hr in 10% EDTA RT, 102 and 73 hr in 10% EDTA 37C, 13.5 and 4.3 hr in 5% nitric acid, and 140 and 36 hr in 10% formic acid, respectively. Decalcification in 10% EDTA at 37C was accelerated, but 10% EDTA at RT provided optimal results for immunohistochemistry and cellular and structural details. Decalcification using 5% nitric acid was accomplished in the shortest time and exhibited good cellular and architectural morphology, whereas 10% formic acid was suboptimal with respect to tissue and cellular morphology. Despite being the slowest method, EDTA at RT is still the recommended method for decalcifying mineralized tissues; however, if rapid decalcification is needed, 5% nitric acid is the best option, yielding acceptable tissue integrity and speed

A New Automated Histomorphometric MATLAB Algorithm for Immunohistochemistry Analysis Using Whole Slide Imaging

The use of animal models along with the employment of advanced and sophisticated stereological methods for assessing bone quality combined with the use of statistical methods to evaluate the effectiveness of bone therapies has made it possible to investigate the pathways that regulate bone responses to medical devices. Image analysis of histomorphometric measurements remains a time-consuming task, as the image analysis software currently available does not allow for automated image segmentation. Such a feature is usually obtained by machine learning and with software platforms that provide image-processing tools such as MATLAB. In this study, we introduce a new MATLAB algorithm to quantify immunohistochemically stained critical-sized bone defect samples and compare the results with the commonly available Aperio Image Scope Positive Pixel Count (PPC) algorithm. Bland and Altman analysis and Pearson correlation showed that the measurements acquired with the new MATLAB algorithm were in excellent agreement with the measurements obtained with the Aperio PPC algorithm, and no significant differences were found within the histomorphometric measurements. The ability to segment whole slide images, as well as defining the size and the number of regions of interest to be quantified, makes this MATLAB algorithm a potential histomorphometric tool for obtaining more objective, precise, and reproducible quantitative assessments of entire critical-sized bone defect image data sets in an efficient and manageable workflow

Poly(2-allylamidopropyl-2-oxazoline)-Based Hydrogels: From Accelerated Gelation Kinetics to In Vivo Compatibility in a Murine Subdermal Implant Model

A rapid photo-curing system based on poly(2-ethyl-2-oxazoline-co-2-allylamidopropyl-2-oxazoline) and its in vivo compatibility are presented. The base polymer was synthesized from the copolymerization of 2-ethyl-2-oxazoline (EtOx) and the methyl ester containing 2-methoxycarboxypropyl-2-oxazoline (C(3)MestOx) followed by amidation with allylamine to yield a highly water-soluble macromer. We showed that spherical hydrogels can be obtained by a simple waterin-oil gelation method using thiol-ene coupling and investigated the in vivo biocompatibility of these hydrogel spheres in a 28-day murine subdermal model. For comparison, hydrogel spheres prepared from poly(ethylene glycol) were also implanted. Both materials displayed mild, yet typical foreign body responses with little signs of fibrosis. This is the first report on the foreign body response of a poly(2-oxazoline) hydrogel, which paves the way for future investigations into how this highly tailorable class of materials can be used for implantable hydrogel devices

Convergence of scaffold-guided bone reconstruction and surgical vascularization strategies—a quest for regenerative matching axial vascularization

The prevalent challenge facing tissue engineering today is the lack of adequate vascularization to support the growth, function, and viability of tissue engineered constructs (TECs) that require blood vessel supply. The research and clinical community rely on the increasing knowledge of angiogenic and vasculogenic processes to stimulate a clinically-relevant vascular network formation within TECs. The regenerative matching axial vascularization approach presented in this manuscript incorporates the advantages of flap-based techniques for neo-vascularization yet also harnesses the in vivo bioreactor principle in a more directed “like for like” approach to further assist regeneration of the specific tissue type that is lost, such as a corticoperiosteal flap in critical sized bone defect reconstruction

Bone Regeneration Exploiting Corticoperiosteal Tissue Transfer for Scaffold-Guided Bone Regeneration

Contemporary reconstructive approaches for critical size bone defects carry significant disadvantages. As a result, clinically driven research has focused on the development and translation of alternative therapeutic concepts. Scaffold-guided tissue regeneration (SGTR) is an emerging technique to heal critical size bone defects. However, issues synchronizing scaffold vascularization with bone-specific regenerative processes currently limit bone regeneration for extra large (XL, 19 cm) critical bone defects. To address this issue, we developed a large animal model that incorporates a corticoperiosteal flap (CPF) for sustained scaffold neovascularization and bone regeneration. In 10 sheep, we demonstrated the efficacy of this approach for healing medium (M, 9 cm) size critical bone defects as demonstrated on plain radiography, microcomputed tomography, and histology. Furthermore, in two sheep, we demonstrate how this approach can be safely extended to heal XL critical size defects. This article presents an original CPF technique in a well-described preclinical model, which can be used in conjunction with the SGTR concept, to address challenging critical size bone defects in vivo. This article describes a novel scaffold-guided tissue engineering approach utilizing a corticoperiosteal flap for bone healing in critical size long bone defects. This approach will be of use for tissue engineers and surgeons exploring vascularized tissue transfer as an option to regenerate large volumes of bone for extensive critical size bone defects both in vivo and in the clinical arena. </p

The Patenting and Technological Trends in Hernia Mesh Implants

Described as a projection (prolapse) of tissue through a fascial defect in the abdominal wall, hernias are associated with significant rates of complications, recurrence, and reoperations. This literature review is aimed at providing an overview of the prosthetic surgical meshes used for the repairing of hernia defects. The review was carried out using two specialized online databases: Espacenet, from the European Patent Office (EPO), and WIPO from the World Intellectual Property Organization. Of the 56 patents selected from 2008 to 2018, China was the largest contributor with 55% (31 patents) of the total patent applicant filings, followed by the United States of America (US), with 29% (16 patents). Although the majority of patent applications (39 documents) had at least one company (industry) assigned to the patent application, 4 patents were solely from academic research. Our data showed that only 13 industry applicants have had their products included in the market, and the majority of meshes available on the market are still made from polypropylene. Chemical, physical, and mesh surface modifications have been implemented, and a few reviews describing mesh design, composition, and mechanical properties are available. However, to date, the ideal mesh implant from a clinical point of view has not been developed.</p