Objectives. The lack of effective treatment for cartilage defects has prompted investigations using tissue engineering techniques for their regeneration and repair. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to a scaffold. Our aim in this study was to repair full-thickness defects in articular cartilage in the weight-bearing area of a porcine model, and to investigate whether the CD44 monoclonal antibody biotin-avidin (CBA) binding technique could provide satisfactory tissue-engineered cartilage. Methods. Cartilage defects were created in the load-bearing region of the lateral femoral condyle of mini-type pigs. The defects were repaired with traditional tissue-engineered cartilage, tissue-engineered cartilage constructed with the biotin-avidin (BA) technique, tissue-engineered cartilage constructed with the CBA technique and with autologous cartilage. The biomechanical properties, Western blot assay, histological findings and immunohistochemical staining were explored. Results. The CBA group showed similar results to the autologous group in biomechanical properties, Moran’s criteria, histological tests and Wakitani histological scoring. Conclusions. These results suggest that tissue-engineered cartilage constructed using the CBA technique could be used effectively to repair cartilage defects in the weight-bearing area of joints. Cite this article: H. Lin, J. Zhou, L. Cao, H. R. Wang, J. Dong, Z. R. Chen. Tissue-engineered cartilage constructed by a biotin-conjugated anti-CD44 avidin binding technique for the repairing of cartilage defects in the weight-bearing area of knee joints in pigs. Bone Joint Res 2017;6:–295. DOI: 10.1302/2046-3758.65.BJR-2016-0277

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 tissue-engineered ligament was designed and evaluated after ACL reconstruction in a rabbit model. Method: Rabbits received tissue-engineered ligaments or tissue-engineered ligaments seeded with primary rabbit ACL cells. The tissue-engineered ligaments were composed of multifilament poly-L-lactide yarn (70 denier) fabricated into novel 24 yarn 3-D braids. Scaffolds were designed to be easily handled and fixed by the surgeon in ACL reconstructions using the suture over the button technique. A continuous scaffold design accommodated the flexibility of intra-articular loads and the rigours of the bone tunnels. The contralateral legs were used as controls. A key parameter for tissue ingrowth was scaffold porosity at 58 ± 9% and mode pore diameter of 183 ± 83 μm. Results: Histological evaluations showed slow collagen tissue infiltration at the surface of the replacement at the four-week time point for both the tissue-engineered ligament and cell-seeded tissue-engineered ligament. At the 12-week time point, both replacements showed collagen ingrowth and remodelling across the entire implant occurred with a thin fibrous capsule. The cell-seeded tissue-engineered ligament demonstrated greater levels of mature collagen ingrowth and healing compared to the non-cell seeded tissue-engineered ligament. The initial tensile strength properties of the scaffold were 332 ± 20 N and 354 ± 68 MPa, which compared well to the rabbit ACL control (314 ± 66 N). The tensile properties of the tissue-engineered ligament and seeded tissue-engineered ligament at four weeks were 67% and 76%, respectively of control. The tensile properties of the biodegradable implant decreased with time for the tissue-engineered and cell seeded tissue-engineered ligament and by 12 weeks was 9% and 30% respectively, as compared to the rabbit ACL control. The 30% strength retention for the tissue-engineered ligament replacements at 12 weeks was greater than reported by others using poly(lactic acid) and polypropylene ligament augmentation devices (LAD) at 12 weeks, with values of 13% and 16% of control strength retention, respectively. Conclusion: The results of this study demonstrate the promise of a novel cell seeded tissue-engineered ligament for anterior cruciate ligament regeneration

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, tissue-engineered substitutes will be developed to serve as functional replacements for total tendon ruptures that closely resemble the original tissue, restoring functionality. Method. Water borne polyurethanes (WBPU) containing acrylate groups, specifically polyethylene glycol methacrylate (PEGMA) or 2-hydroxyethyl methacrylate (HEMA), were combined with mouse mesenchymal stem cells (MoMSCs) and heparin sodium to formulate bioinks for the fabrication of scaffolds via extrusion-based 3D bioprinting. Result. The biocompatibility of acrylated-WBPUs was confirmed in 2D with MoMSCs using lactate dehydrogenase assay, DNA assay and live/dead assays. Cell-laden scaffolds were 3D-bioprinted by encapsulating MoMSCs at varying cell densities within the acrylated WBPUs. The resulting 3D structures support cell viability and proliferation within the scaffolds, as confirmed by live/dead assay, lactate dehydrogenase assay and DNA assays. Differentiation studies in the 3D-bioprinted scaffolds demonstrated the phenotype transition of MoMSCs toward tenocytes through gene expression and protein deposition analysis. The inclusion of sodium heparin in the bioinks revealed increased synthesis of matrix assembly proteins within the 3D-bioprinted constructs. Conclusion. The developed bioinks were biocompatible and printable, supporting cell viability within the 3D-bioprinted scaffold. The fabricated cell-laden constructs sustained cell proliferation, differentiation, and tissue formation. The addition of heparin sodium enhanced tissue formation and organization, showing promising results for the regeneration of tendon total ruptures. Principio del formularioThis work was supported by the Spanish State Research Agency (AEI) under grant No CPP2021-008754. The authors would like to thank their partners in the project, which are in charge of the synthesis of heparin sodium and acrylated-WBPUs

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 tissue-engineered tendons were used to reconstruct the ACL in mini-pigs and mechanical strength was assessed after three months. Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and GAG, and the tensile strength of recellularized tendons. After in vivo reconstruction, the tensile strength of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the native strength of the ACL. Our translational study indicates that the recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and mechanical strength of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts

Objectives. To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects. Materials and Methods. Bone marrow-derived, autologous MSCs were seeded on Acropora or Porites coral granules in a perfusion bioreactor. Acropora-TECs (n = 7), Porites-TECs (n = 6) and bone autografts (n = 2) were then implanted into 25 mm long metatarsal diaphyseal defects in sheep. Bimonthly radiographic follow-up was completed until killing four months post-operatively. Explants were subsequently processed for microCT and histology to assess bone formation and coral bioresorption. Statistical analyses comprised Mann-Whitney, t-test and Kruskal–Wallis tests. Data were expressed as mean and standard deviation. Results. A two-fold increaseof newly formed bone volume was observed for Acropora-TECs when compared with Porites-TECs (14 . sd. 1089 mm. 3. versus 782 . sd. 507 mm. 3. ; p = 0.09). Bone union was consistent with autograft (1960 . sd. 518 mm. 3. ). The kinetics of bioresorption and bioresorption rates at four months were different for Acropora-TECs and Porites-TECs (81% . sd. 5% versus 94% . sd. 6%; p = 0.04). In comparing the defects that healed with those that did not, we observed that, when major bioresorption of coral at two months occurs and a scaffold material bioresorption rate superior to 90% at four months is achieved, bone nonunion consistently occurred using coral-based TECs. Discussion. Bone regeneration in critical-size defects could be obtained with full bioresorption of the scaffold using coral-based TECs in a large animal model. The superior performance of Acropora-TECs brings us closer to a clinical application, probably because of more suitable bioresorption kinetics. However, nonunion still occurred in nearly half of the bone defects. Cite this article: A. Decambron, M. Manassero, M. Bensidhoum, B. Lecuelle, D. Logeart-Avramoglou, H. Petite, V. Viateau. A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model. Bone Joint Res 2017;6:208–215. DOI: 10.1302/2046-3758.64.BJR-2016-0236.R1

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 tissue-engineered tendons to the clinic. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The purpose of this work was to create an in vitro model of tissue-engineered cartilage structure produced by isolated swine articular chondrocytes, expanded in culture and seeded onto a biological scaffold. Swine articular chondrocytes were enzymatically isolated from pig joints and expanded in monolayer culture. When confluence was reached, cells were resuspended and seeded in vitro onto biological collagen scaffolds for 3, 4 and 6 weeks. Samples were retrieved from the culture and analysed macroscopically and biomechanically by compressive test. Gross evaluation was performed by simple probing, sizing and weighing the samples at all time periods. A baseline of the values was also recorded at time 0. Then, samples were biomechanically tested by unconfined compression and shear tests. Finally, the samples were fixed in 4% paraformaldehyde and processed for histological evaluation. Some samples were stained with Safranin-o, and some others subjected to immunostaining analysis for type II collagen. Upon retrieval, samples showed dimensional enlargement and mass increase over time and gross mechanic integrity by simple probing. A biomechanical test demonstrated an initial reduction in the values of compressive and shear parameters, followed by a consistent increase throughout the tested time points. Histology showed cartilage-like tissue maturing over time within the biological scaffold. The results from this study demonstrate that isolated chondrocytes could be seeded onto a biological collagen scaffold, producing cartilage-like matrix with tissue-specific morphology and biomechanical integrity. This tissue-engineered cartilage structure is easily reproducible and it could represent a valuable model for studying the behaviour of different variables on the newly formed cartilage

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.

Acknowledgements: This work was undertaken as part of the UK Research and Innovation (UKRI)-funded CDT in Advanced Biomedical Materials.

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 tissue-engineered composite presents cartilaginous appearance and biomechanical integrity after 9 weeks in vivo

Autogenous bone grafting limitations have motivated the development of Tissue-Engineered (TE) biomaterials that offer an alternative as bone void fillers. However, the lack of a blood supply within implanted constructs may result in avascular necrosis and construct failure. 1. The aim of this project was to investigate the potential of novel TE constructs to promote vascularisation and bone defect repair using two distinct approaches. In Study 1, we investigated the potential of a mesenchymal stem cell (MSC) and endothelial cell (EC) co-culture to stimulate pre-vascularisation of biomaterials prior to in vivo implantation. 2. In Study 2, we investigated the potential of TE hypertrophic cartilage to promote the release of angiogenic factors such as VEGF, vascular invasion and subsequent endochondral bone formation in an in vivo model. Collagen-only (Coll), collagen-glycosaminoglycan (CG) and collagen-hydroxyapatite (CHA) scaffolds were fabricated by freeze-drying. 3. , seeded with cells and implanted into critical-sized calvarial and femoral defects in immunocompetent rats. In Study 1, Coll and CG scaffolds were initially seeded with ECs, allowed to form capillary-like networks before the delayed addition of MSCs and continued culture prior to calvarial implantation. In Study 2, CG and CHA scaffolds were seeded with MSCs and cultured under chondrogenic and subsequent hypertrophic conditions to form a cartilage pre-cursor prior to calvarial and femoral implantation in vivo. MicroCT and histomorphometry quantification demonstrated the ability of both systems to support increased bone formation compared to controls. Moreover, the greatest levels of bone formation were observed in the CG groups, notably in those containing cartilage tissue (Study 2). Assessment of the immune response suggests the addition of MSCs promotes the polarisation of macrophages away from inflammation (M1) towards a pro-remodelling phenotype (M2). We have developed distinct collagen-based systems that promote vascularisation and ultimately enhance bone formation, confirming their potential as advanced strategies for bone repair applications

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.

In vivo study showed that bone regeneration was highly improved in presence of pBMP-2. Micro-CT shows a filling of the gyroid sinuses of the implant (Figure 1).

In vitro, the presence of BMP2 did not influence the viability of the osteoblasts and the mortality remained below 3%. After 7 days, the presence of BMP2 in the scaffold significantly increased by 85 and 65% the COL1A1 expression and by 8 and 33-fold the TNAP expression by osteoblasts in the monolayer or in the scaffold, respectively. This BMP2 effect was transient in monolayer and did not modify gene expression at day 14.

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.

Introduction

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.

Summary

Nasal Chondrocytes are safe and feasible for tissue engineering approaches in articular cartilage repair.

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 (COLII/Sox9/Aggrecan) and reduced expressions of COLI and Runx2 (osteogenic) than static group. However, the higher expression of COLX in the perfused constructs suggests a shear stress role in AC hypertrophy. Both conditions (perfused/static) stained positively for GAG deposition and for the presence of collagen II and aggrecan. Overall, the results provide a proof-of-concept of our customizable extruded bioreactor envisaging applications as a platform for AC-TE repair strategies and in the development of more reliable in vitro models for disease modelling and drug screening.

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 in vitro expansion of bone graft for subsequent implantation and investigated the optimal conditions for growing new mineralised bone in vitro.

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 tissue-engineered replacement to be successful, a comprehensive understanding of the complete anatomy of the native structure is essential. Knowledge of the dimensions of ligament footprints is vitally important for surgeons as it guides the placement of bone tunnels during repair. It is also imperative in tissue-engineered design as the creation of a successful replacement relies on a thorough understanding of the native anatomy and microanatomical structure. Several studies explore techniques to describe ligament footprints around the body, with limited studies describing in-depth footprint dimensions of the ATFL, CFL and PTFL. Techniques currently used to measure ligament footprints are complex and require resources which may not be readily available, therefore a new methodology may prove beneficial. Objectives. This study explores the application of a novel technique to assess the footprint of ankle ligaments through a straightforward inking method. This method aims to enhance surgical technique and contribute to the development of a tissue-engineered analogue based on real anatomical morphometric data. Methods. Cadaveric dissection of the ATFL, CFL and PTFL was performed on 12 unpaired fresh frozen ankles adhering to regulations of the Human Tissue (Scotland) Act. The ankle complex with attaching ligaments was immersed in methylene blue. Dissection of the proximal and distal entheses of each ligament was carried out to reveal the unstained ligament footprint. Images of each ligament footprint were taken, and the area, length and width of each footprint were assessed digitally. Results. The collective area of the proximal entheses of the ATFL, CFL and PTFL measures 142.11 ± 12.41mm2. The mean areas of the superior (SB) and inferior band (IB) of the distal enthesis of the ATFL measured 41.72 ± 5.01mm2 and 26.66 ± 3.12mm2 respectively. The footprint of the distal enthesis of the CFL measured 146.07 ± 14.05mm2, while the footprint of the distal PTFL measured 126.26 ± 8.88mm2. The proximal footprint of the ATFL, CFL and PTFL measured 11.06 ± 0.69mm, 7.87 ± 0.43mm and 10.52 ± 0.63mm in length and 8.66 ± 0.50mm, 9.10 ± 0.92mm and 14.41 ± 1.30mm in width on average. The distal footprint of the ATFL (SB), ATFL (IB), CFL and PTFL measured 10.92 ± 0.81 mm, 8.46 ± 0.46mm, 13.98 ± 0.93mm and 11.25 ± 0.95mm in length and 7.76 ± 0.59mm, 7.51 ± 0.64mm, 18.98 ± 1.15mm and 24.80 ± 1.25mm in width on average. Conclusions. This methodology provides an effective approach in the identification of the footprint of the lateral ligaments of the ankle to enhance surgical precision and accuracy in tissue-engineered design. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

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 tissue-engineered VBPC showed great capability in promoting angiogenesis and osteogenesis in vivo. It may provide a novel approach to create a superior blood supply and nutritional environment to overcome the deficits of current artificial bone graft substitutes. Cite this article: Bone Joint Res 2023;12(12):722–733

It is well known that environmental cues such as mechanical loading and/or cell culture medium composition affect tissue-engineered constructs resembling natural bone. These studies are mostly based on an initial setting of the influential parameter that will not be further changed throughout the study. Through the growth of the cells and the deposition of the extracellular matrix (ECM) the initial environmental conditions of the cells will change, and with that also the loads on the cells will change. This study investigates how changes of mechanical load or media composition during culture influences the differentiation and ECM production of mesenchymal stromal cells seeded on porous 3D silk fibroin scaffolds. ECM formation, ECM mineralization and cell differentiation in 3D tissue-engineered bone were analyzed using microscopic tools. Our results suggest that mechanical stimuli are necessary to differentiate human mesenchymal stromal cells of both bone marrow and adipose tissue origin into ECM producing osteoblasts which ultimately become ECM-embedded osteocytes. However, the influence of this stimulus seems to fade quickly after the onset of the culture. Constructs which were initially cultured under mechanical loading continued to deposit minerals at a similar growth rate once the mechanical stimulation was stopped. On the other hand, cell culture medium supplementation with FBS was identified as an extremely potent biochemical cue that influences the mechanosensitivity of the cells with regards to cell differentiation, ECM secretion and mineral deposition. Only through a thorough understanding on these influences over time will we be able to predictably control tissue development in vitro

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This given study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or gas fluorination combined with the foam (F+coll). Cell free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, biomechanical properties, cell vitality and content, histopathology of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed. Implantation of the scaffolds did not negatively affect mice weight development and organs, indicating biocompatibility. All scaffolds maintained their size and shape for the duration of the implantation. A high cell viability was detected in the scaffolds prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties when compared to the controls. Mild signs of inflammation (foreign-body giant cells, hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable in vivo, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in the in vivo compared to the samples cultured 1 week in vitro, but did not differ between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. (shared first authorship). Acknowledgement: The study was supported by DFG grants SCHU1979/9-1 and SCHU1979/14-1

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 tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering