Jellyfish collagens exhibit auspicious perspectives for tissue engineering applications primarily due to their outstanding compatibility with a wide range of cell types, low immunogenicity and biodegradability. Furthermore, derived from a non-mammalian source, jellyfish collagens reduce the risk of disease transmission, minimising therefore the ethical and safety concerns. The current study aims to investigate the potential of 3-dimensional jellyfish collagen sponges (3D-JCS) in promoting bone tissue regeneration. Both qualitative and quantitative analyses were performed in order to assess adhesion and proliferation of MC3T3 cells on 3D-JCL, as well as cell migration and bone-like ECM production. Histological and fluorescent dyes were used to stain mineral deposits (i.e. Alizarin Red S (ARS), Von Kossa, Tetracycline hydrochloride) while images were acquired using optical and confocal microscopy. Qualitative data indicated successful adhesion and proliferation of MC3T3 cells on the 3D-JCS as well as cell migration along with ECM production both on the inner and outer surface of the scaffolds. Moreover, quantitative analyses indicated a four-fold increase of ARS uptake between 2- and 3-dimensional cultures (N=3) as well as an eighteen-fold increase of ARS uptake for the 3D-JCS (N=3) when cultured in osteogenic conditions compared to control. This suggests the augmented osteogenic potential of MC3T3 cells when cultured on 3D-JCS. Nevertheless, the cell-mediated mineral deposition appeared to alter the mechanical properties of the jellyfish collagen sponges that were previously reported to exhibit low mechanical properties (compressive modulus: 1-2 kPa before culture). The biocompatibility, high porosity and pore interconnectivity of jellyfish collagen sponges promoted adhesion and proliferation of MC3T3 cells as well as cell migration and bone-like ECM production. Their unique features recommend the jellyfish collagen sponges as superior biomaterial scaffolds for bone tissue regeneration. Further studies are required to quantify the change in mechanical properties of the cell-seeded scaffolds and confirm their suitability for bone tissue regeneration. We predict that the 3D-JCS will be useful for future studies in both bone and bone-tendon interface regeneration. Acknowledgments. This research has been supported by a Medical Research Scotland Studentship award (ref: -50177-2019) in collaboration with Jellagen Ltd

Bone fractures are highly observed clinical situation in orthopaedic treatments. In some cases, there might be non-union problems. Therefore, recent studies have focused on tissue engineering applications as alternative methods to replace surgical procedures. Various biopolymer based scaffolds are produced using different fabrication techniques for bone tissue engineering applications. In this study, hydroxyapatite (HAp) and loofah containing carboxymethyl chitosan (CMC) scaffolds were prepared. In this regard, first 4 ml of CMC solution, 0.02 g of hydroxyapatite (HAP) and 0.06 g of poly (ethylene glycol) diglycidyl ether (PEGDE) were mixed in an ultrasonic bath until the HAp powders were suspended. Next, 0.04 g of loofah was added to the suspension and with the help of PEGDE as the cross-linking agent, then, the mixture was allowed to cross-link at 40. o. C overnight. Finally, the three-dimensional, porous and sponge-like scaffolds were obtained after lyophilization (TELSTAR - LyoQuest −85) at 0.1 mbar and −25°C for 2 days. Morphologies, chemical structures and thermal properties of the scaffolds were characterized by scanning electron microscopy (SEM), Fourier Transform infrared spectroscopy (FT-IR) and thermogravimetric differential thermal analysis (TGA/DTA), respectively. In addition, swelling behavior and mechanical properties of the scaffolds under compression loading were determined. In order to investigate biocompatibility of the scaffolds, WST-1 colorimetric assay at days 0, 1, 3, 5 and 7 was conducted by using human dermal fibroblast. Also, histological and morphological analysis were performed for cell attachment at day 7. In conclusion, the produced scaffolds showed no cytotoxic effect. Therefore, they can be considered as a candidate scaffold for bone tissue regeneration. Further studies will be performed by using bone marrow and periosteum derived mesenchymal stem cells with these scaffolds

Bone tissue engineering is a promising strategy to treat the huge number of bone fractures caused by progressive population ageing and diseases i.e., osteoporosis. The bioactive and biomimetic materials design modulating cell behaviour can support healthy bone tissue regeneration. In this frame, type I collagen and hydroxyapatite (HA) have been often combined to produce biomimetic scaffolds. In addition, mesoporous bioactive glasses (MBGs) are known for their ability to promote the deposition of HA nanocrystals and their potential to incorporate and release therapeutic ions. Furthermore, the use of 3D printing technologies enables the effective design of scaffolds reproducing the natural bone architecture. This study aims to design biomimetic and bioactive 3D printed scaffolds that mimic healthy bone tissue natural features in terms of chemical composition, topography and biochemical cues. Optimised collagenous hybrid systems will be processed by means of extrusion 3D printing technologies to obtain high resolution bone-like structures. Protocols of human co-cultures of osteoblasts and osteoclasts will be developed and used to test the 3D scaffolds. Type I collagen has been combined with rod-like nano-HA and strontium containing MBGs (micro- and nano-sized particles) in order to obtain hybrid systems resembling the composition of native bone tissue. A comprehensive rheological study has been performed to investigate the potential use of the hybrid systems as biomaterial inks. Mesh-like structures have been obtained by means of extrusion-based technologies exploiting the freeform reversible embedding of suspended hydrogels (FRESH) approach. Different crosslinking methods have been tested to improve final constructs mechanical properties. Both crosslinked and non-crosslinked biomaterials were cultured with human osteoblasts and osteoclasts to assay the hybrid matrix biocompatibility as well as its influence on cell behaviour. Homogeneous hybrid systems have been successfully developed and characterised, proving their suitability as biomaterial inks for 3D printing technologies. Mesh-like structures have been extruded in a thermo-reversible gelatine slurry, exploiting the sol-gel transition of the systems under physiological conditions. Covalent bonds between collagen molecules have been promoted by genipin treatment, leading to a significant increase in matrix strength and stability. The collagen methacrylation and the further UV-crosslinking are under investigation as alternative promising method to reinforce the 3D structure during the printing process. Biological tests showed the potential of the developed systems especially for genipin treated samples, with a significant adhesion of primary cells. Collagenous hybrid systems proved their suitability for bioactive 3D printed structures design for bone tissue engineering. The multiple stimuli provided by the scaffold composition and structure will be investigated on both direct and indirect human osteoblasts and osteoclasts co-culture, according to the developed protocols

In therapeutic bone repairs, autologous bone grafts, conventional or vascularized allografts, and biocompatible artificial bone substitutes all have their shortcomings. Tissue engineering may be an alternative for cranial bone repair. Titanium (Ti) and its alloys are widely used in many clinical devices because of perfect biocompatibility, highly corrosion resistance and ideal physical properties. An important progress in treating bone defects has been the introduction of bone morphogenetic proteins (BMPs), specifically BMP-2. The proteins induce osteogenic cell differentiation in vitro, as well as bone defect healing in vivo. In this study, we fabricated the titanium plate with dioxide creating by microarc oxidation (MAO) and then electronic deposition of Ca.P that can carrier recombinant human bone morphogenetic protein-2 (rhBMP-2) to enhance osteogenesis in vitro and bone formation in vivo. The rhBMP-2 was controlled released from MAO-Ca.P-rhBMP2 implant was maintain within 35days longer than Ti without MAO modification group and without CaP electronic deposition group. In addition, the in vitro results revealed that the bioactivity of rhBMP-2 released from MAO-Ca.P-rhBMP2 implant with an ideal therapeutic dose was well maintained. In vivo, the critical-sized defect (20-mm diameter) of New Zealand White rabbits was used to experiment. We concluded that sustained controlled-release of rhBMP-2 above a therapeutic dose could induce osseointegration between the implant and surrounding bone the rate of bone formation into the implant and produce neovascularization. Our study combined the concept of osteoconductive and osteoinductive to do the bone tissue regeneration

Bone grafts are crucial for the treatment of bone defects caused by tumor excision. The gold standard is autograft but their availability is limited. Allografts are an alternative, but there is a risk of rejection by the immune system. The tissue engineering field is trying to develop vascularized bone grafts, using innovative biomaterials for surgery applications. While the gold standard in bone graft in dentistry is the use of decellularized bovine bone particles (Bio-Oss®), our work has produced a polysaccharide-based composite matrix (composed of PUllulan, DextraNand particles of HydroxyApatite (PUDNHA), as a new scaffold for promoting bone formation and vascularization of the tissue. In the context of bone tissue regeneration, the function of osteoblast and endothelial cells has been extensively studied, while the impact of osteocytes has been regarded as secondary. Nonetheless, the osteocytes represent 90–95% of bone cells and are responsible for orchestration of bone remodeling. Here, we propose an original method to analyze the interaction between bone and biomaterials, after in vivo implantation of the matrix PUDNHA in an experimental sheep model. Our objectives are to analyze the network established by osteocytes in the newly formed tissue induced by the matrix, as well as their interactions with the blood vessels. Sheep have been implanted with the Bio-Oss® or the PUDNHA using the sinus lift technique. After 3 (3M) and 6 months (6M), the animals were euthanazied and the explants were fixed, analyzed by X-ray, embedded in Methylmetacrylate/Buthylmetacrylate and analyzed histologically by Trichrome staining. Thereafter, the samples (n=3/group) were polished using different sand papers. A final polish was realized using a 1µm Diamond polishing compound. The blocks were incubated 10 or 30s with 37% phosphoric acid to remove the mineral on the surface, then dipped in 2.6% sodium hypochlorite to remove the collagen. The samples were air dried overnight, metallized with Gold palladium the following day, before being imaged with a SEM. As expected, PUDNHA activates bone regeneration in this sinus lift model after 3M and 6M. X-ray analysis and histological data revealed more bone regeneration at 6M versus 3M in both groups. With this acid eching technique, we were able to visualize the interface of bone with the biomaterials. This treatment coupled with SEM analysis, confirmed the increase of bone formation with time of implantation in both groups. In addition, SEM images revealed that osteocyte alignment and their network were different in the new regenerated bone compared to the host bone. Moreover, images showed the direct contact of the osteocytes with the blood vessels formed in the new regenerated bone. This acid eching technique can be useful in the field of biomaterials to see the relationship between cells, blood vessels and the material implanted and understand how the new bone is forming around the different biomaterials

Worldwide 500,000 cases of maxillofacial cancer are diagnosed each year. After surgery, the reconstruction of large bone defect is often required. The induced membrane approach (Masquelet, 2000) is one of the strategies, but exhibits limitations in an oncological context (use of autografts with or without autologous cells and Bone Morphogenetic Proteins). The objectives of this work are to develop an injectable osteoinductive and osteoconductive composite matrix composed of doped strontium (Sr) hydroxyapatite (HA) particles dispersed within a polysaccharide scaffold, to evaluate in vitro their ability to stimulate osteoblastic differentiation of human mesenchymal stem cells (hMSC) and to stimulate in vivo bone tissue regeneration. HA particles were synthesized with different ratios of Sr. X-ray diffraction (XRD), Inductively Coupled Plasma (ICP), and particle size analysis (Nanosizer™) were used to characterize these particles. HA and Sr-doped HA were dispersed at different ratios within a pullulan-dextran based matrices (Autissier, 2010), Electronic scanning microscopy Back Scattering Electron microscopy (ESEM-BSE) and ICP were used to characterize the composite scaffolds. In vitro assays were performed using hMSC (cell viability using Live/Dead assay, expression of osteoblastic markers by quantitative Polymerase Chain Reaction). Matrices containing these different particles were implanted subcutaneously in mice and analyzed by Micro-Computed Tomography (micro-CT) and histologically (Masson's trichrome staining) after 2 and 4 weeks of implantation. XRD analysis was compatible with a carbonated hydroxyapatite and patterns of Sr-doped HA are consistent of Sr substitution on HA particles. Morphological evaluation (TEM and Nanosizer™) showed that HA and Sr-doped HA particles form agglomerates (150 nm to 4 µm). Matrices composed with different ratios of HA or Sr-doped-HA, exhibit a homogenous distribution of the particles (ESEM-BSE), whatever the conditions of substitution. In vitro studies revealed that Sr-doped HA particles within the matrix stimulates the expression of osteoblastic markers, compared to non-doped HA matrices. Subcutaneous implantation of the matrices demonstrated the formation of a mineralized tissue. Quantitative analyses show that the mineralization of the implants is dependent of the amount of HA particles dispersed, with an optimal ratio of 5% of particles. Histological analysis revealed osteoid tissue in contact to the matrix. In conclusion, the ability of this injectable composite scaffold to promote ectopically tissue mineralization is promising for bone tissue engineering. Osseous implantation in a femoral bone defect in rats is now in progress. 5% of doped HA particles were implanted within the induced membranes in a context of radiotherapy procedure. Micro-CT analyses are ongoing. This new matrix could represent an alternative to the autografts for the regeneration of large bone defects in an oncological context

Summary Statement. A coupled finite element - analytical model is presented to predict and to elucidate a clinical healing scenario where bone regenerates in a critical-sized femoral defect, bounded by periosteum or a periosteum substitute implant and stabilised via an intramedullary nail. Introduction. Bone regeneration and maintenance processes are intrinsically linked to mechanical environment. However, the cellular and subcellular mechanisms of mechanically-modulated bone (re-) generation are not fully understood. Recent studies with periosteum osteoprogenitor cells exhibit their mechanosensitivity in vitro and in situ. In addtion, while a variety of growth factors are implicated in bone healing processes, bone morphogenetic protein-2 (BMP-2) is recognised to be involved in all stages of bone regeneration. Furthermore, periosteal injuries heal predominantly via endochondral ossification mechanisms. With this background in mind, the current study aims to understand the role of mechanical environment on BMP-2 production and periosteally-mediated bone regeneration. The one-stage bone transport model [1] provides a clinically relevant experimental platform on which to model the mechanobiological process of periosteum-mediated bone regeneration in a critical-sized defect. Here we develop a model framework to study the cellular-, extracellular- and mechanically-modulated process of defect infilling, governed by the mechanically-modulated production of BMP-2 by osteoprogenitor cells located in the periosteum. Methods. Material properties of the healing callus and periosteum contribute to the strain stimulus sensed by osteoprogenitor cells therein. Using a mechanical finite element model, periosteal surface strains are first predicted as a function of callus properties. Strains are then input to a mechanistic mathematical model, where mechanical regulation of BMP-2 production mediates rates of cellular proliferation, differentiation and extracellular matrix (ECM) production, to predict healing outcomes. A parametric approach enables the spatial and temporal prediction of tissue regeneration via endochondral ossification. Predictions are compared with experimental, micro-computed tomographic and histologic, measures of cartilage and mineralised bone tissue regenerates. Model Predictions in Light of Experimental Case Studies: A validated baseline model predicts defect healing via cellular egression, extracellular matrix production and endochondral ossification, using parameters optimised to mimic experimental outcome measures at initial and final stages of healing. To elucidate which predictive model paramenters result in the intrinsic differences in experimental outcomes between defects bounded by either periosteum in situ or a periosteum substitute implant, model parameters are then varied by orders of magnitude to determine which factors exert dominant influence on achievement of experimentally relevant ECM area outcomes. Considering the complete set of parameters relevant to healing, the rate of osteoprogenitor to osteoblast differentiation, as well as rates of chondrocyte and osteoblast proliferation must be reduced and ECM production by chondrocytes must be increased from baseline, to achieve healing outcomes analogous to those observed in experiments. Discussion/Conclusion. The novel model framework presented here integrates a mechanistic feedback system, based on the mechanosensitivity of periosteal osteoprogenitor cells, which allows for modeling and prediction of tissue regeneration on multiple length and time scales

Stromal cells derived from human dental pulp (HDPSCs) are of current interest for applications in skeletal tissue engineering. Angiogenesis and revascularization of bone grafts or bone constructs in vivo are of paramount importance for bone tissue regeneration and/or fracture healing. The aim of this study was to investigate the angiogenic and osteogenic potential of HDPSCs in combination with Bioglass¯ scaffolds in vitro and in vivo. HDPSCs, isolated by collagenase digestion, were either maintained as monolayers or dynamically seeded on 3D Bioglass¯ scaffolds and cultured under either basal or osteogenic conditions for 2 and 4 weeks. Expression of osteogenic (COL1A1, ALP, RUNX2 and OC) and angiogenic markers (VEGFR2, CD34 and PECAM1) was determined using qRT-PCR. Alternatively, constructs were either cultured in vitro under basal/osteogenic conditions for 6 weeks or sealed in diffusion chambers which were then implanted intraperitoneally in immunosuppressed mice for 8 weeks. Retrieved constructs were fixed and embedded for histology and immunohistochemistry using antibodies against COL1, RUNX2, OC, VEGFR2, CD34 and PECAM1. qRT-PCR showed no significant differences in gene expression of osteogenic markers between basal and osteogenic media for both 3D construct and monolayers. However when comparing 3D constructs to monolayers: COL1A1 showed a significantly lower expression (p< 0.05) in 3D compared to 2D at 2 weeks in both culture conditions, and this pattern was reversed after 4 weeks. ALPL was significantly lower in 3D constructs at 2 weeks under both conditions (p<0.01), and was significantly higher in basal conditions at 4 weeks (p<0.05). RUNX2 showed higher expression in 3D constructs at all time points and under both conditions while OC showed lower expression in 3D constructs at 2 weeks and higher expression at 4 weeks under both conditions. For the angiogenic markers, 3D constructs under osteogenic conditions showed an increase of expression in VEGFR2 and PECAM1 at 2 weeks followed by a decrease at week 4, while CD34 expression was undetected in 3D constructs at all times and under both sets of culture conditions. The expression of VEGFR2 and PECAM 1 under both conditions and at both time points was greater in 3D constructs compared to monolayers. After 8 weeks, the in vivo retrieved constructs showed no signs of inflammatory reactions. Immunohistology confirmed positive staining of osteogenic and angiogenic markers in 3D constructs from both in vitro and in vivo experiments with a greater staining intensity seen in the in vivo constructs. Furthermore, the in vivo constructs showed more intense sirius red staining and higher intensity of immunostaining using antibodies to type 1 collagen, with higher calcification as indicated by alizarin red staining. In conclusion, this study indicated that a combination of HDPSCs and Bioglass¯ scaffolds has potential to provide a suitable microenvironment for angiogenic and osteogenic differentiation of HDPSCs which is essential for bone regeneration in preclinical and/or clinical applications

Introduction. The annual incidence of fractures in the UK is almost 4%. Bone grafting procedures and segmental bone transport have been employed for bone tissue regeneration. However, their limited availability, donor site morbidity and increased cost mean that there is still a large requirement for alternative methods and there is considerable research into regeneration using bone morphogenetic proteins (BMPs). The aims of this study are to synthesise and combine BMP-2 with a novel nanocomposite and study its release. Materials and Methods. BMP-2 was synthesised using an E. coli expression system and purified. C2C12 cells were used to test its bioactivity using an alkaline phosphatase (ALP) assay. The modified solution evaporation method was used to fabricate 30% a-TCP/PLGA nanocomposite and it was characterized using SEM, TEM, TGA, XRD, EDX and particle size analysis. The release pattern of adsorbed BMP-2 was studied using an ELISA assay. Results. SEM suggests that there was a homogeneous distribution of a-TCP nanoparticles within the PLGA matrix. The concentration of BMP-2 adsorbed onto a-TCP/PLGA nanocomposites directly correlated with the incubation concentration of BMP-2. Approximately 10-15% of BMP-2 was adsorbed on to the discs, up to an incubation concentration of 25 μg/ml. At a higher incubation concentration (50 μg/ml), however, only 4% of the BMP-2 appears to have been adsorbed. The ALP activity shows that the BMP-2 was bioactive and successfully adsorbed onto the surface of the a-TCP/PLGA nanocomposite. A burst release pattern of BMP-2 was observed over 24h, being maximal at 2 h. Discussion. Increasing incubation concentrations of BMP-2 resulted in an increase of detected adsorbed BMP-2 on the discs, however this was not observed at the highest incubation concentration (50 μg/ml). As adsorption of BMP-2 onto the ground surface of the a-TCP/PLGA nanocomposite occurs primarily through electrostatic interactions between cationic BMP-2 and anionic a-TCP, this might reflect saturation in adsorption secondary to saturation of surface anionic a-TCP by BMP-2, or heterogeneity of the discs' content and/or surface area. Adsorbed BMP-2 was shown to have bioactivity which significantly increased with increasing incubation concentrations of BMP-2 and suggests this nanocomposite could have osteoinductive potential in-vivo. The burst pattern of BMP-2 release has been shown previously from BMP adsorbed onto mPCL/collagen/HA composite and this significantly increased the bone formation of critical-sized defects. Whilst a more sustained release profile of BMP-2 is generally considered desirable, this nanocomposite of a-TCP/PLGA has been shown to possess some osteoconductive and weak osteoinductive properties itself (unpublished). The addition of BMP-2 to the nanocomposite by adsorption results in an early burst release, which can promote the differentiation of mesenchymal cells into osteoblasts. The proliferation of these might then be sustained by the nanocomposite itself, without the need for sustained delivery of BMP-2. Conclusions. Bioactive BMP-2 was synthesised and combined with a-TCP/PLGA nanocomposite, producing a biodegradable and osteoinductive material which has potential for use in bone regeneration

Objectives

There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite.