Objectives. The lack of effective treatment for cartilage defects has prompted investigations using tissue engineering techniques for their regeneration and repair. The success of
Introduction and Aims: There are more than 200,000 anterior cruciate ligament (ACL) ruptures each year in the United States. The replacements used for ACL repair do not fully recreate the ACL’s function and histological appearance. Therefore, a novel
Introduction. Tendon ruptures represent one of the most common acute tendon injuries in adults worldwide, affecting millions of people anually and becoming more prevalent due to longer life expectancies and sports activities. Current clinical treatments for full tears are unable to completely restore the torn tendons to their native composition, structure and mechanical properties. To address this clinical challenge,
Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful, autografts or allografts have certain disadvantages, and synthetic grafts have had poor clinical results. The aim of this study was to determine the efficacy of tissue engineering decellularized tibialis tendons by recellularization and culture in a dynamic tissue bioreactor. To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow-derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to hose experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes, and newly synthesized collagen and glycosaminoglycan (GAG) were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The mechanical strength of recellularized constructs was measured after dynamic stimulation. Finally, the
Objectives. To compare the therapeutic potential of
Abstract. Objectives. Musculoskeletal injuries are the leading contributor to disability globally, yet current treatments do not offer complete restoration of the tissue. This has resulted in the exploration of novel interventions based on tissue engineering as a therapeutic solution. This study aimed to explore novel collagen sponges as scaffolds for bone tissue engineering as an initial step in the construction of tendon-bone co-culture constructs in vitro. Methods. Collagen sponges (Jellagen, UK), manufactured from Jellyfish collagen were seeded with 10,000 rat osteoblast cells (dROBs) and maintained in culture for 6 days (37°C, 5% CO. 2. ). Qualitative viability was assessed by a fluorescent Calcein-AM live cell stain and quantitively via the CYQUANT cell viability assay (Invitrogen, UK) on days 0, 1, 4 and 6 in culture (n=3 per time point). Digital imaging was also used to assess size and shape changes to the collagen sponge in culture. Results. The collagen sponge biomaterial supported dROB adhesion, viability and proliferation with an abundance of viable cells detected by fluorescent microscopy on day 6. Indeed, the quantitative assessment confirmed that cellular proliferation was evident with increases in fluorescence detected from 517 (± 88) RFU to 8730 ± (2228) RFU from day 0 to 6. In addition, the size of the collagen sponges appeared to decrease over time, indicating contraction of the collagen sponges in culture. Conclusions. This preliminary study has demonstrated that the novel collagen sponges support cellular attachment and proliferation of osteoblasts, and is an important first step in building a bone-tendon construct in vitro. Our future work is focussed on using the osteoblast-seeded sponges in combination with tendon cells, to build a co-culture to represent the bone-tendon interface in vitro. This work has the potential to advance the clinical translation of
The purpose of this work was to create an in vitro model of
Meniscal injuries affect over 1.5 million people across Europe and the USA annually. Injury greatly reduces knee joint mobility and quality of life and frequently leads to the development of osteoarthritis. Tissue engineered strategies have emerged in response to a lack of viable treatments for meniscal pathologies. However, to date, constructs mimicking the structural and functional organisation of native tissue, whilst promoting deposition of new extracellular matrix, remains a bottleneck in meniscal repair. 3D bioprinting allows for deposition and patterning of biological materials with high spatial resolution. This project aims to develop a biomimetic 3D bioprinted meniscal substitute. Meniscal tissue was characterised to effectively inform the design of biomaterials for bioprinting constructs with appropriate structural and functional properties. Histology, gene expression and mass spectrometry were performed on native tissue to investigate tissue architecture, matrix components, cell populations and protein expression regionally across the meniscus. 3D laser scanning and magnetic resonance imaging were employed to acquire the external geometrical information prior to fabrication of a 3D printed meniscus. Bioink suitability was investigated through regional meniscal cell encapsulation in blended hydrogels, with the incorporation of growth factors and assessed for their suitability through rheology, scanning electron microscopy, histology and gene expression analysis. Meniscal tissue characterisation revealed regional variations in matrix compositions, cellular populations and protein expression. The process of imaging through to 3D printing highlighted the capability of producing a construct that accurately replicated meniscal geometries. Regional meniscal cell encapsulation into hydrogels revealed a recovery in cell phenotype, with the incorporation of growth factors into the bioink's stimulating cellular re-differentiation and improved zonal functionality. Meniscus biofabrication highlights the potential to print patient specific, customisable meniscal implants. Achieving zonally distinct variations in cell and matrix deposition highlights the ability to fabricate a highly complex tissue engineered construct.
The purpose of this study was to assess the physical, biochemical and biomechanical properties of a cartilage matrix-chondrocyte-fibrin glue composite as biological tool for cartilage repair. Chondrocytes were enzymatically isolated from pig joints and resuspended in fibrinogen solution. Articular cartilage was harvested from pig joints, chopped into small chips and lyophiliaed. Cartilage chips were rehydrated and mixed with the cell/fibrinogen solution and with thrombin, in order to form a fibrin glue gel composite with cells and chips (group A). Control composites were made from lyophilised cartilage chips assembled with fibrin glue, but not containing chondrocytes (group B). Other control groups included fibrin glue/chondrocyte specimens without cartilage chips (group C) and specimens made of the fibrin glue alone (group D). All samples were weighed and implanted into subcutaneous pouches of nude mice. Animals were sacrificed at 2 and 9 weeks. Samples were evaluated grossly and the final/initial mass ratio was calculated. Samples were evaluated histologically, biomechanically, and biochemically. Upon retrieval, only the samples in experimental group A retained their original pre-implantation mass. Histological analysis showed newly formed cartilage matrix in the specimens from group A and C. Biomechanical analysis showed significantly higher modulus in experimental samples, with respect to the other groups at the latest time point. Analysis of hydraulic permeability showed significantly decreasing values for all groups throughout the experimental times and lowest values for the experimental samples of group A in the latest time point, although there was no statistically significant difference among the groups. Biochemical analysis demonstrated higher values in the latest time point for samples prepared with cells for water and GAG content, whereas highest values for hydroxyproline were recorded for samples assembled with cartilage chips. DNA analysis showed higher values of samples prepared with chondrocytes and fibrin glue and also an important increase in values of the samples made of fibrin glue only, indicating a possible host fibroblast growth inside the samples over time. This
Autogenous bone grafting limitations have motivated the development of
To design slow resorption patient-specific bone graft whose properties of bone regeneration are increased by its geometry and composition and to assess it in in-vitro and in-vivo models. A graft composed by hydroxyapatite (HA) and β-TCP was designed as a cylinder with 3D gyroid porosities and 7 mm medullary space based on swine's anatomy. It was produced using a stereolithography 3D-printing machine (V6000, Prodways). Sterile bone grafts impregnated with or without a 10µg/mL porcine BMP-2 (pBMP-2) solution were implanted into porcine femurs in a bone loss model. Bone defect was bi-weekly evaluated by X-ray during 3 months. After sacrifice, microscanner and non-decalcified histology analysis were conducted on biopsies. Finally, osteoblasts were cultured inside the bone graft or in monolayer underneath the bone graft. Cell viability, proliferation, and gene expression were assessed after 7 and 14 days of cell culture (n=3 patients). 3D scaffolds were successfully manufactured with a composition of 80% HA and 20% β-TCP ±5% with indentation compressive strength of 4.14 MPa and bending strength of 11.8MPa.
BMP2-impregnated bone graft is a promising patient-personalized 3D-printed solution for bone defect regeneration, by promoting neighboring host cells recruitment and solid new bone formation. For any figures and tables, please contact the authors directly.
Endochondral ossification (EO) is the process of bone development via a cartilage template. It involves multiple stages, including chondrogenesis, mineralisation and angiogenesis. Importantly, how cartilage mineralisation affects angiogenesis during EO is not fully understood. Here we aimed to develop a new in vitro co-culture model to recapitulate and study the interaction between mineralised cartilage generated from human mesenchymal stromal cells (hMSCs) and microvascular networks. Chondrogenic hMSC pellets were generated by culture with transforming growth factor (TGF)-β3. For mineralised pellets, β-glycerophosphate (BGP) was added from day 7 and TGF-β3 was withdrawn on day 14. Conditioned medium (CM) from the pellets was used to evaluate the effect on human umbilical vein endothelial cells (HUVECs) in migration, proliferation and tube formation assays. To perform direct co-cultures, pellets were embedded in fibrin hydrogels containing vessel-forming cells (HUVECs, adipose stromal cells) for 10 days with BGP to induce mineralisation. The pellets and hydrogels were characterised by immunohistochemistry and confocal imaging.Introduction
Method
Nasal Chondrocytes are safe and feasible for tissue engineering approaches in articular cartilage repair. As compared to articular chondrocytes (AC), nasal septum chondrocytes (NC) proliferate faster and have a higher and more reproducible capacity to generate hyaline-like cartilaginous tissues. Moreover, the use of NC would allow reducing the morbidity associated with the harvesting of cartilage biopsy from the patient. The objective of the present study was to demonstrate safety and feasibility in the use of tissue engineered cartilage graft based on autologous nasal chondrocytes for the repair of articular defect in goats.Summary
Introduction
Bioreactors have been used in articular cartilage tissue engineering (AC-TE) to apply different mechanical stimuli in an attempt to better mimic the native AC microenvironment. However, these systems are often highly complex, costly and not very versatile. In this work, we propose a simple and customizable perfusion bioreactor fabricated by 3D-extrusion to study the effect of shear stress in human bone-marrow mesenchymal stem cells (hBMSC) cultured in 3D porous polycaprolactone (PCL) scaffolds. Prototype models were designed in a CAD-software to perfectly fit the scaffolds and computational fluid dynamics analysis was used to predict the fluid velocities and shear stress forces inside the bioreactor. For the culture studies, hBMSC-PCL constructs were cultured under static expansion for 2 weeks and then transferred to the ABS-extruded bioreactors for continuous perfusion culture (0.2mL/min) under chondrogenic induction for additional 3 weeks. Perfused constructs showed similar cell proliferation and higher sGAG production in comparison to the static counterparts (bioreactor without perfusion). Constructs exposed to shear stress stimuli presented higher expressions of chondrogenic genes (
Nanoscale topography increases the bioactivity of a material and stimulates specific responses (third generation biomaterial properties) at the molecular level upon first generation (bioinert) or second generation (bioresorbable or bioactive) biomaterials. We developed a technique (based upon the effects of nanoscale topography) that facilitated the Two topographies (nanopits and nanoislands) were embossed into the bioresorbable polymer Polycaprolactone (PCL). Three dimensional cell culture was performed using double-sided embossing of substrates, seeding of both sides, and vertical positioning of substrates. The effect of Hydroxyapatite, and chemical stimulation were also examined. Human bone marrow was harvested from hip arthroplasty patients, the mesenchymal stem cells culture expanded and used for cellular analysis of substrate bioactivity. The cell line specificity and osteogenic behaviour was demonstrated through immunohistochemistry, confirmed by real-time PCR and quantitative PCR. Mineralisation was demonstrated using alizarin red staining. Results showed that the osteoinduction was optimally conferred by the presence of nanotopography, and also by the incorporation of hydroxyapatite (HA) into the PCL. The nanopit topography and HA were both superior to the use of BMP2 in the production of mineralised bone tissue. The protocol from shim production to bone marrow harvesting and vertical cell culture on nanoembossed HaPCL has been shown to be reproducible and potentially applicable to economical larger scale production.
Abstract. The lateral ligaments of the ankle composed of the anterior talofibular (ATFL), calcaneofibular (CFL) and posterior talofibular ligaments (PTFL), are amongst the most commonly injured ligaments of the human body. Although treatment methods have been explored exhaustively, healing outcomes remain poor with high rates of re-injury, chronic ankle instability and pain persisting. The introduction and application of tissue engineering methods may target poor healing outcomes and eliminate long-term complications, improving the overall quality of life of affected individuals. For any surgical procedure or
Aims. Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration. Methods. A total of 24 male New Zealand white rabbits were divided into four groups according to the experimental materials. Allogenic adipose-derived mesenchymal stem cells (AMSCs) were cultured and seeded evenly in the collagen/chitosan sheet to form cell sheet as periosteum. Simultaneously, allogenic AMSCs were seeded onto alginate beads and were cultured to differentiate to endothelial-like cells to form vascularized bone construct (VBC). The cell sheet was wrapped onto VBC to create a vascularized bone-periosteum construct (VBPC). Four different experimental materials – acellular construct, VBC, non-vascularized bone-periosteum construct, and VBPC – were then implanted in bilateral L4-L5 intertransverse space. At 12 weeks post-surgery, the bone-forming capacities were determined by CT, biomechanical testing, histology, and immunohistochemistry staining analyses. Results. At 12 weeks, the VBPC group significantly increased new bone formation volume compared with the other groups. Biomechanical testing demonstrated higher torque strength in the VBPC group. Notably, the haematoxylin and eosin, Masson’s trichrome, and immunohistochemistry-stained histological results revealed that VBPC promoted neovascularization and new bone formation in the spine fusion areas. Conclusion. The
It is well known that environmental cues such as mechanical loading and/or cell culture medium composition affect
Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction,
Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a