Optimisation and validation of a custom-designed perfusion bioreactor for bone tissue engineering: Flow assessment and optimal culture environmental conditions

Various perfusion bioreactor systems have been designed to improve cell culture with three-dimensional porous scaffolds, and there is some evidence that fluid force improves the osteogenic commitment of the progenitors. However, because of the unique design concept and operational configuration, the experimental setups of perfusion bioreactor systems are not always compatible. To reconcile results from different systems, the thorough optimisation and validation of the experimental configuration are required in each system. In this study, optimal experimental conditions for a perfusion bioreactor were explored in 3 steps. First, an in silico modelling was performed using a scaffold geometry obtained by microCT and an expedient geometry parameterised with porosity and permeability to assess the accuracy of calculated fluid shear stress and computational time. Then, environmental factors for cell culture were optimised, including the volume of the medium, bubble suppression, and medium evaporation. Further, by combining the findings, it was possible to determine the optimal flow rate at which cell growth was supported but osteogenic differentiation was triggered. Here, we demonstrated that fluid shear stress, ranging from nearly 0 to 15 mPa, was sufficient to induce osteogenesis, but cell growth was severely impacted by the volume of perfused medium, the presence of air bubbles, and medium evaporation, all of which are common concerns in perfusion bioreactor systems. This study emphasises the necessity of optimisation of experimental variables, which may often be underreported or overlooked, and indicates steps which can be taken to address issues common to perfusion bioreactors for bone tissue engineering.publishedVersio

Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model

Background: In bone tissue engineering (BTE), extensive research into vascular endothelial growth factor A (VEGFA)-mediated angiogenesis has yielded inconsistent results. The aim of this study was to investigate the influence on angio- and osteogenesis of adenoviral-mediated delivery of VEGFA alone or in combination with bone morphogenetic protein 2 (BMP2) in bone marrow stromal cells (BMSC) seeded onto a recently developed poly(LLA-co-CL) scaffold. Methods: Human BMSC were engineered to express VEGFA alone or in combination with BMP2 and seeded onto poly(LLA-co-CL) scaffolds. Changes in angiogenic and osteogenic gene and protein levels were examined by quantitative reverse-transcription polymerase chain reaction (RT-PCR), PCR array, and alkaline phosphatase assay. An in vivo subcutaneous mouse model was used to investigate the effect on angio- and osteogenesis of VEGFA alone or in combination with BMP2, using microcomputed tomography (μCT), histology, immunohistochemistry, and immunofluorescence. Results: Combined delivery of a lower ratio (1:3) of VEGFA and BMP2 (ad-BMP2 + VEGFA) led to upregulation of osteogenic and angiogenic genes in vitro at 3 and 14 days, compared with mono-delivery of VEGFA (ad-VEGFA) and other controls. In vivo, in a subcutaneous mouse model, both ad-VEGFA and ad-BMP2 + VEGFA scaffold explants exhibited increased angiogenesis at 2 weeks. Enhanced angiogenesis was largely related to the recruitment and differentiation of mouse progenitor cells to the endothelial lineage and, to a lesser extent, to endothelial differentiation of the implanted BMSC. μCT and histological analyses revealed enhanced de novo bone formation only in the ad-BMP2 + VEGFA group, corresponding at the molecular level to the upregulation of genes related to osteogenesis, such as ALPL, RUNX2, and SPP1. Conclusions: Although BMSC expressing VEGFA alone or in combination with BMP2 significantly induced angiogenesis, VEGFA alone failed to demonstrate osteogenic activity both in vitro and in vivo. These results not only call into question the use of VEGFA alone in bone regeneration, but also highlight the importance in BTE of appropriately formulated combined delivery of VEGFA and BMP2.publishedVersio

Understanding of how the properties of medical grade lactide based copolymer scaffolds influence adipose tissue regeneration: Sterilization and a systematic in vitro assessment

Aliphatic polyesters are the synthetic polymers most commonly used in the development of resorbable medical implants/devices. Various three-dimensional (3D) scaffolds have been fabricated from these polymers and used in adipose tissue engineering. However, their systematic evaluation altogether lacks, which makes it difficult to select a suitable degradable polymer to design 3D resorbable implants and/or devices able to effectively mimic the properties of adipose tissue. Additionally, the impact of sterilization methods on the medical devices, if any, must be taken into account. We evaluate and compare five different medical-grade resorbable polyesters with l-lactide content ranging from 50 to 100 mol% and exhibiting different physiochemical properties depending on the comonomer (d-lactide, ε-caprolactone, glycolide, and trimethylene carbonate). The salt-leaching technique was used to prepare 3D microporous scaffolds. A comprehensive assessment of physical, chemical, and mechanical properties of the scaffolds was carried out in PBS at 37 °C. The cell-material interactions and the ability of the scaffolds to promote adipogenesis of human adipose tissue-derived stem cells were assessed in vitro. The diverse physical and mechanical properties of the scaffolds, due to the different composition of the copolymers, influenced human adipose tissue-derived stem cells proliferation and differentiation. Scaffolds made from polymers which were above their glass transition temperature and with low degree of crystallinity showed better proliferation and adipogenic differentiation of stem cells. The effect of sterilization techniques (electron beam and ethylene oxide) on the polymer properties was also evaluated. Results showed that scaffolds sterilized with the ethylene oxide method better retained their physical and chemical properties. Overall, the presented research provides (i) a detailed understanding to select a degradable polymer that has relevant properties to augment adipose tissue regeneration and can be further used to fabricate medical devices/implants; (ii) directions to prefer a sterilization method that does not change polymer properties.publishedVersio

Understanding of how the properties of medical grade lactide based copolymer scaffolds influence adipose tissue regeneration: Sterilization and a systematic in vitro assessment

Aliphatic polyesters are the synthetic polymers most commonly used in the development of resorbable medical implants/devices. Various three-dimensional (3D) scaffolds have been fabricated from these polymers and used in adipose tissue engineering. However, their systematic evaluation altogether lacks, which makes it difficult to select a suitable degradable polymer to design 3D resorbable implants and/or devices able to effectively mimic the properties of adipose tissue. Additionally, the impact of sterilization methods on the medical devices, if any, must be taken into account. We evaluate and compare five different medical-grade resorbable polyesters with l-lactide content ranging from 50 to 100 mol% and exhibiting different physiochemical properties depending on the comonomer (d-lactide, ε-caprolactone, glycolide, and trimethylene carbonate). The salt-leaching technique was used to prepare 3D microporous scaffolds. A comprehensive assessment of physical, chemical, and mechanical properties of the scaffolds was carried out in PBS at 37 °C. The cell-material interactions and the ability of the scaffolds to promote adipogenesis of human adipose tissue-derived stem cells were assessed in vitro. The diverse physical and mechanical properties of the scaffolds, due to the different composition of the copolymers, influenced human adipose tissue-derived stem cells proliferation and differentiation. Scaffolds made from polymers which were above their glass transition temperature and with low degree of crystallinity showed better proliferation and adipogenic differentiation of stem cells. The effect of sterilization techniques (electron beam and ethylene oxide) on the polymer properties was also evaluated. Results showed that scaffolds sterilized with the ethylene oxide method better retained their physical and chemical properties. Overall, the presented research provides (i) a detailed understanding to select a degradable polymer that has relevant properties to augment adipose tissue regeneration and can be further used to fabricate medical devices/implants; (ii) directions to prefer a sterilization method that does not change polymer properties

Efficacy of treating segmental bone defects through endochondral ossification: 3D printed designs and bone metabolic activities

Three-dimensional printing (3D printing) is a promising technique for producing scaffolds for bone tissue engineering applications. Porous scaffolds can be printed directly, and the design, shape and porosity can be controlled. 3D synthetic biodegradable polymeric scaffolds intended for in situ bone regeneration must meet stringent criteria, primarily appropriate mechanical properties, good 3D design, adequate biocompatibility and the ability to enhance bone formation. In this study, healing of critical-sized (5 ​mm) femur defects of rats was enhanced by implanting two different designs of 3D printed poly(l-lactide-co-ε-caprolactone) (poly(LA-co-CL)) scaffolds seeded with rat bone marrow mesenchymal stem cells (rBMSC), which had been pre-differentiated in vitro into cartilage-forming chondrocytes. Depending on the design, the scaffolds had an interconnected porous structure of 300–500 ​μm and porosity of 50–65%. According to a computational simulation, the internal force distribution was consistent with scaffold designs and comparable between the two designs. Moreover, the defects treated with 3D-printed scaffolds seeded with chondrocyte-like cells exhibited significantly increased bone formation up to 15 weeks compared with empty defects. In all experimental animals, bone metabolic activity was monitored by positron emission tomography 1, 3, 5, 7, 11 and 14 weeks after surgery. This demonstrated a time-dependent relationship between scaffold design and metabolic activity. This confirmed that successful regeneration was highly reproducible. The in vitro and in vivo data indicated that the experimental setups had promising outcomes and could facilitate new bone formation through endochondral ossification.publishedVersio

Ectopic Bone Tissue Engineering in Mice Using Human Gingiva or Bone Marrow-Derived Stromal/Progenitor Cells in Scaffold-Hydrogel Constructs

Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4–8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC (p 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups (p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications

Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial

Background Autologous grafting, despite some disadvantages, is still considered the gold standard for reconstruction of maxillofacial bone defects. The aim of this study was to evaluate bone regeneration using bone marrow-derived mesenchymal stromal cells (MSCs) in a clinical trial, a less invasive approach than autologous bone grafting. This comprehensive clinical trial included subjects with severe mandibular ridge resorption. Methods The study included 11 subjects aged 52–79 years with severe mandibular ridge resorption. Bone marrow cells were aspirated from the posterior iliac crest and plastic adherent cells were expanded in culture medium containing human platelet lysate. The MSCs and biphasic calcium phosphate granules as scaffolds were inserted subperiosteally onto the resorbed alveolar ridge. After 4–6 months of healing, new bone formation was assessed clinically and radiographically, as were safety and feasibility. Bone at the implant site was biopsied for micro-computed topography and histological analyses and dental implants were placed in the newly regenerated bone. Functional outcomes and patient satisfaction were assessed after 12 months. Results The bone marrow cells, expanded in vitro and inserted into the defect together with biphasic calcium phosphate granules, induced significant new bone formation. The regenerated bone volume was adequate for dental implant installation. Healing was uneventful, without adverse events. The patients were satisfied with the esthetic and functional outcomes. No side effects were observed. Conclusions The results of this comprehensive clinical trial in human subjects confirm that MSCs can successfully induce significant formation of new bone, with no untoward sequelae. Hence, this novel augmentation procedure warrants further investigation and may form the basis of a valid treatment protocol, challenging the current gold standard

The educational value of ward rounds as a learning and teaching opportunity for house officers, medical officers, and registrars in Sudanese hospitals: a multi-center cross-sectional study

Abstract Background Ward rounds are a cornerstone in the educational experience of junior doctors and an essential part of teaching patient care. Here, we aimed to assess the doctors’ perception of ward rounds as an educational opportunity and to identify the obstacles faced in conducting a proper ward round in Sudanese hospitals. Method A cross-sectional study was conducted from the 15th to the 30th of January 2022 among house officers, medical officers, and registrars in about 50 teaching and referral hospitals in Sudan. House officers and medical officers were considered the learners, while specialist registrars were considered the teachers. Doctors’ perceptions were assessed using an online questionnaire, with a 5-level Likert scale to answer questions. Results A total of 2,011 doctors participated in this study (882 house officers, 697 medical officers, and 432 registrars). The participants were aged 26.9 ± 3.2 years, and females constituted about 60% of the sample. An average of 3.1 ± 6.8 ward rounds were conducted per week in our hospitals, with 11.1 ± 20.3 h spent on ward rounds per week. Most doctors agreed that ward rounds are suitable for teaching patient management (91.3%) and diagnostic investigations (89.1%). Almost all the doctors agreed that being interested in teaching (95.1%) and communicating appropriately with the patients (94.7%) make a good teacher in ward rounds. Furthermore, nearly all the doctors agreed that being interested in learning (94.3%) and communicating appropriately with the teacher (94.5%) make a good student on ward rounds. About 92.8% of the doctors stated that the quality of ward rounds could be improved. The most frequently reported obstacles faced during ward rounds were the noise (70%) and lack of privacy (77%) in the ward environment. Conclusion Ward rounds have a special value in teaching patient diagnosis and management. Being interested in teaching/learning and having good communication skills were the two major criteria that make a good teacher/learner. Unfortunately, ward rounds are faced with obstacles related to the ward environment. It is mandatory to ensure the quality of both ward rounds' teaching and environment to optimize the educational value and subsequently improve patient care practice