Introduction. Degenerative meniscal tears are the most common meniscal lesions, representing huge clinical and socio-economic burdens. Their role in knee osteoarthritis (OA) onset and progression is well established and demonstrated by several retrospective studies. Effective preventive measures and non-surgical treatments for degenerative meniscal lesions are still lacking, also because of the lack of specific and accurate animal models in which test them. Thus, we aim to develop and validate an accurate animal model of meniscus degeneration. Method. Three different surgical techniques to induce medial meniscus degenerative changes in ovine model were performed and compared. A total of 32 sheep (stifle joints) were subjected to either one of the following surgical procedures: a) direct arthroscopic mechanical meniscal injury; b) peripheral devascularization and denervation of medial meniscus; c) full thickness medial femoral condyle cartilage lesion. In all the 3 groups, the contralateral joint served as a control. Result. From a visual examination of the knee joint emerged a clear difference between control and operated groups, in the menisci but also in the cartilage, indicating the onset of OA-related cartilage degeneration. The meniscal and cartilaginous lesions were characterized by different severity and location in the different groups. For instance, a direct meniscal injury caused cartilaginous lesions especially in the medial part of the condyles, and the other approaches presented specific signature. Evaluation of scoring scales (e.g. ICRS score) allowed the quantification of the damage and the identification of differences among the four groups. Conclusion. We were effectively able to develop and validate a sheep model of meniscal degeneration which led to the onset of OA. This innovative model will allow to test in a pre-clinical relevant setting innovative approaches to prevent meniscal-related OA. Funding. Project PNRR-MAD-2022-12375978 funded by Italian Ministry of Health

Objectives. The treatment of osteoporotic fractures is a major challenge, and the enhancement of healing is critical as a major goal in modern fracture management. Most osteoporotic fractures occur at the metaphyseal bone region but few models exist and the healing is still poorly understood. A systematic review was conducted to identify and analyse the appropriateness of current osteoporotic metaphyseal fracture animal models. Materials and Methods. A literature search was performed on the Pubmed, Embase, and Web of Science databases, and relevant articles were selected. A total of 19 studies were included. Information on the animal, induction of osteoporosis, fracture technique, site and fixation, healing results, and utility of the model were extracted. Results. Fracture techniques included drill hole defects (3 of 19), bone defects (3 of 19), partial osteotomy (1 of 19), and complete osteotomies (12 of 19). Drill hole models and incomplete osteotomy models are easy to perform and allow the study of therapeutic agents but do not represent the usual clinical setting. Additionally, biomaterials can be filled into drill hole defects for analysis. Complete osteotomy models are most commonly used and are best suited for the investigation of therapeutic drugs or noninvasive interventions. The metaphyseal defect models allow the study of biomaterials, which are associated with complex and comminuted osteoporotic fractures. Conclusion. For a clinically relevant model, we propose that an animal model should satisfy the following criteria to study osteoporotic fracture healing: 1) induction of osteoporosis, 2) complete osteotomy or defect at the metaphysis unilaterally, and 3) internal fixation. Cite this article: R. M. Y. Wong, M. H. V. Choy, M. C. M. Li, K-S. Leung, S. K-H. Chow, W-H. Cheung, J. C. Y. Cheng. A systematic review of current osteoporotic metaphyseal fracture animal models. Bone Joint Res 2018;7:6–11. DOI: 10.1302/2046-3758.71.BJR-2016-0334.R2

Objectives. We studied subchondral intraosseous pressure (IOP) in an animal model during loading, and with vascular occlusion. We explored bone compartmentalization by saline injection. Materials and Methods. Needles were placed in the femoral condyle and proximal tibia of five anaesthetized rabbits and connected to pressure recorders. The limb was loaded with and without proximal vascular occlusion. An additional subject had simultaneous triple recordings at the femoral head, femoral condyle and proximal tibia. In a further subject, saline injections at three sites were carried out in turn. Results. Loading alone caused a rise in subchondral IOP from 11.7 mmHg (. sd. 7.1) to 17.9 mmHg (. sd. 8.1; p < 0.0002). During arterial occlusion, IOP fell to 5.3 mmHg (. sd. 4.1), then with loading there was a small rise to 7.6 mmHg (. sd. 4.5; p < 0.002). During venous occlusion, IOP rose to 20.2 mmHg (. sd. 5.8), and with loading there was a further rise to 26.3 mmHg (. sd. 6.3; p < 0.003). The effects were present at three different sites along the limb simultaneously. Saline injections showed pressure transmitted throughout the length of the femur but not across the knee joint. Conclusion. This is the first study to report changes in IOP in vivo during loading and with combinations of vascular occlusion and loading. Intraosseous pressure is not a constant. It is reduced during proximal arterial occlusion and increased with proximal venous occlusion. Whatever the perfusion state, in vivo load is transferred partly by hydraulic pressure. We propose that joints act as hydraulic pressure barriers. An understanding of subchondral physiology may be important in understanding osteoarthritis and other bone diseases. Cite this article: M. Beverly, S. Mellon, J. A. Kennedy, D. W. Murray. Intraosseous pressure during loading and with vascular occlusion in an animal model. Bone Joint Res 2018;7:511–516. DOI: 10.1302/2046-3758.78.BJR-2017-0343.R2

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine. Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2

Summary. Osteoporosis reduces particle-induced osteolysis in rat model. Introduction. Wear particle induced osteolysis is considered to be a vital factor that reduces the life span of joint prosthesis. Osteoporosis is not rare in patients with indication for arthroplasty. However, the influence of osteoporosis on wear particles induced osteolysis is not clear. This study is aimed to explore on this issue by using animal model. Methods. 42 female Sprague-Dawley (SD) rats aged 6 months were randomly divided into 3 groups: A, B and C group. Group A and B contained 18 rats each, and group C contained 6 rats. The rats in group A underwent bilateral ovariectomy. Group B was normal control, and group C was sham control. After 3 months, 6 rats in group A, 6 rats in group B and all the rats of group C were sacrificed. Bone mineral density (BMD), μCT and bone histomorphometry were conducted. The rest of rats in group A were randomly divided into 2 groups: group A1 and group A2, and so were the rats in group B. 5mg titanium particles were implanted onto the calvaria of groups A1 and B1, and isometric PBS solution were injected to group A2 and B2. Calvaria were harvested after 14 days. Calvaria were analyzed by μCT and histomorphometry to measure the osteolysis area of calvarial sagittal suture. Results. Compared with B and C group, BMD and bone histomorphometry index of group A was significantly reduced (P<0.05), and tibial trabeculae of group A was slimmer. Area of calvarial sagittal suture osteolysis were 0.262±0.009mm. 2. , 0.130±0.013mm. 2. , 0.307±0.013mm. 2. and 0.178±0.011mm. 2. in A1, A2, B1and B2 groups, respectively. There was significant difference among the groups. Conclusions. Osteoporosis may reduce particle-induced osteolysis in rat model

The biomechanical evaluation of tendon repair with collagen-based scaffolds in rat model is a common method to determine the functional outcome of the tested material. We introduced a magnetic resonance imaging (MRI) approach to verify the biomechanical test data. In present study different collagen scaffolds for tendon repair were examined. Two collagen test materials: based on bovine stabilized collagen, chemically cross-linked with oriented collagenous fibres (material 1) and based on porcine dermal extracellular matrix, with no cross-linking (material 2) were compared. The animal study was approved by the local review board. Surgery was performed on male Sprague-Dawley rats with a body weight of 400 ± 19 g. Each rat underwent a 5 mm transection of the right Achilles tendon. The M. plantaris tendon was removed. The remaining tendon ends were re-joined with a 5 mm scaffold of either the material 1 or 2. Each scaffold material was sutured into place with two single stiches (Vicryl 4–0, Ethicon) each end. A total of 16 rats (n= 8 each group) were observed for 28 days follow up. The animals were sacrificed and hind limbs were transected proximal to the knee joint. MRI was performed using a 7 Tesla scanner (BioSpec 70/30, Bruker). T2-weighted TurboRARE sequences with an in-plane resolution of 0.12 mm and a slice thickness of 0.7 mm were analysed. All soft and hard tissues were removed from the Achilles tendon-calcaneus-foot complex before biomechanical testing. Subsequently, the specimens were fixed in a materials testing machine (Z1.0, Zwick, Ulm, Germany) for tensile testing. All tendons were preloaded with 1 N and subsequently stretched at a rate of 1 mm/s until complete failure was observed. Non-operated tendons were used as a control (n=4). After 28 postoperative days, MRI demonstrated that four scaffolds (material 1: n=2, material 2: n=2) were slightly dislocated in the proximal part of hind limb. In total five failures of reconstruction could be detected in the tendon repairs (material 1: n=3, material 2: n=2). Tendons augmented with the bovine material 1 showed a maximum tensile load of 57.9 ± 17.9 N and tendons with porcine scaffold material 2 of 63.1 ± 19.5 N. The native tendons demonstrated only slightly higher loads of 76.6 ± 11.6 N. Maximum failure load of the tendon-scaffold construct in both groups did not differ significantly (p < 0.05). Stiffness of the tendons treated with the bovine scaffold (9.9 ± 3.6 N/mm) and with the porcine scaffold (10.7 ± 2.7 N/mm) showed no differences. Stiffness of the native healthy tendon of the contralateral site was significantly higher (20.2 ± 6.6 N/mm, p < 0.05). No differences in the mechanical properties between samples of both scaffold groups could be detected, regardless of whether the repaired tendon defect has failed or the scaffold has been dislocated. The results show that MRI is important as an auxiliary tool to verify the biomechanical outcome of tendon repair in animal models

Long term, secondary implant fixation of Total Disc Replacements (TDR) can be enhanced by hydroxyapatite or similar osseo-conductive coatings. These coatings are routinely applied to metal substrates. The objective of this in vivo study was to investigate the early stability and subsequent bone response adjacent to an all polymer TDR implant over a period of six months in an animal model. Six skeletally mature male baboons (Papio annubis) were followed for a period of 6 months. Using a transperitoneal exposure, a custom-sized Cadisc L device was implanted into the disc space one level above the lumbo-sacral junction in all subjects. Radiographs of the lumbar spine were acquired prior to surgery, and post-operatively at intervals up to 6 months to assess implant stability. Flourochrome markers (which contain molecules that bind to mineralization fronts) were injected at specified intervals in order to investigate bone remodeling with time. Animals were humanely euthanized six months after index surgery. Test and control specimens were retrieved, fixed and subjected to histological processing to assess the bone-implant-bone interface. Fluorescence microscopy and confocal scanning laser microscopy were utilized with BioQuant image analysis to determine the bone mineral apposition rates and gross morphology. Radiographic evaluation revealed no loss of disc height at the operative level or adjacent levels. No evidence of subsidence or significant migration of the implant up to 6 months. Heterotopic ossification was observed to varying degrees at the operated level. Histology revealed the implant primary fixation features embedded within the adjacent vertebral endplates. Flourochrome distribution revealed active bone remodeling occurring adjacent to the polymeric end-plate with no evidence of adverse biological responses. Mineral apposition rates of between 0.7 and 1.7 microns / day are in keeping with literature values for hydroxyapatite coated implants in cancellous sites of various species. Radiographic assessment demonstrates that the Cadisc L implant remains stable in vivo with no evidence of subsidence or significant migration. Histological analysis suggests the primary fixation features are engaged, and in close apposition with the adjacent vertebral bone. Flourochrome markers provide evidence of a positive bone remodelling response in the presence of the implant

Stem cells represent an exciting biological therapy for the management of many musculoskeletal tissues that suffer degenerative disease and/or where the reparative process results in non-functional tissue (‘failed healing’). The original hypothesis was that implanted cells would differentiate into the target tissue cell type and synthesise new matrix. However, this has been little evidence that this happens in live animals compared to the laboratory, and more recent theories have focussed on the immunomodulatory effects via the release of paracrine factors that can still improve the outcome, especially since inflammation is now considered one of the central processes that drive poor tendon healing. Because of the initial ‘soft’ regulatory environment for the use of stem cells in domestic mammals, bone and fat-derived stem cells quickly established themselves as a useful treatment for naturally occurring musculoskeletal diseases in the horse more than 20 years ago (Smith, Korda et al. 2003). Since the tendinopathy in the horse has many similarities to human tendinopathy, we propose that the following challenges and, the lessons learnt, in this journey are highly relevant to the development of stem cells therapies for human tendinopathy:

Introduction. Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy affecting approximately 3 million people in China (Stone R, 2009). The precise aetiology of KBD is not clear, but the lack of selenium and the pollution of mycotoxins in food are a suspected cause of KBD. In this pilot study, we use a rat model to investigate the effect of low selenium and T-2 toxin on articular cartilage metabolism. Methods. 140 male Sprague-Dawley rats were fed with selenium-deficient or normal diet for 4 weeks to produce a low selenium or normal nutrition status. The rats were then fed for a further 4 weeks with low selenium or normal diets with or without T-2 toxin (100ng per gram body weight per day). The rat knee joints were fixed and paraffin embedded and histological and immunohistochemical staining was performed to analyse the metabolism of articular cartilage. Results. There was increased cell cluster formation in the middle and/or deep zones in rats fed with both diets. However, an apparent cell loss was observed in the low selenium + T-2 toxin group with an apparent increase in caspase-3 staining, indicating the increased cell apoptosis. Moreover, toluidine blue staining was reduced in the low selenium + T-2 toxin group, suggesting a loss of sulphated glycosaminoglycans. Similarly, there was reduced 2B6 and 6C3 staining in the territorial matrix of chondrocytes, indicating a reduced synthesis in 4-sulhated and native CS motifs. In contrast, increased 1B5 staining was observed in the articular cartilage from the low selenium + T-2 toxin group, suggesting a lack of CS sulphatransferase activity. Interestingly, there was increased 7D4 staining in the superficial zone of articular cartilage from low selenium + T-2 toxin group, suggesting an initiation of an osteoarthritis-like lesion. Discussion. These results indicated that low selenium nutrition and T-2 toxin could promote cell apoptosis and disrupt CS-GAG metabolism in ECM of rat articular cartilage in this animal model, which is similar to that observed in KBD patients. Collectively, our results support the hypothesis that low selenium and T-2 toxin are the cause of KBD

Introduction

The objective of this study was to investigate the effects of different doses rhBMP-2 on bone healing in an ovine lumbar interbody fusion model.

Summary Statement

Conventional imaging techniques lack the ability to objectively assess early stages of intervertebral disc degeneration, characterised by glycosaminoglycan loss. This study shows that MRI T2∗ mapping correlates positively with GAG content and that it provides continuous measurements for disc degeneration.

INTRODUCTION

In the treatment of nonunions, and other complications of bone repair, an attractive alternative to bone autografts would be the use of a combination of autologous mesenchymal progenitors cells (MSCs), biomaterials and growth factors. Our goal was to determine the therapeutic potential and contribution to the repair process of different sources of mesenchymal stem cells for the treatment of nonunions.

There is little information about the effects of extracorporeal shock-wave about application the effects (ESWA) of on normal bone physiology. We have therefore investigated the effects of ESWA on intact distal rabbit femora in vivo. The animals received 1500 shock-wave pulses each of different energy flux densities (EFD) on either the left or right femur or remained untreated. The effects were studied by bone scintigraphy, MRI and histopathological examination.

Ten days after ESWA (0.5 mJ/mm² and 0.9 mJ/mm² EFD), local blood flow and bone metabolism were decreased, but were increased 28 days after ESWA (0.9 mJ/mm²). One day after ESWA with 0.9 mJ/mm² EFD but not with 0.5 mJ/mm², there were signs of soft-tissue oedema, epiperiosteal fluid and bone-marrow oedema on MRI. In addition, deposits of haemosiderin were found epiperiosteally and within the marrow cavity ten days after ESWA.

We conclude that ESWA with both 0.5 mJ/mm² and 0.9 mJ/mm² EFD affected the normal bone physiology in the distal rabbit femur. Considerable damaging side-effects were observed with 0.9 mJ/mm² EFD on periosteal soft tissue and tissue within the bone-marrow cavity.

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks.

A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres.

We used an in vivo model to assess the use of an autogenous cancellous bone block and marrow graft for augmenting tendon reattachment to metallic implants. We hypothesised that augmentation of the tendon-implant interface with a bone block would enable retention of the graft on the implant surface, enhance biological integration, and result in more consistent functional outcomes compared with previously reported morcellised graft augmentation techniques.

A significant improvement in functional weight-bearing was observed between six and 12 weeks. The significant increase in ground reaction force through the operated limb between six and 12 weeks was greater than that reported previously with morcellised graft augmented reconstructions. Histological appearance and collagen fibre orientation with bone block augmentation more closely resembled that of an intact enthesis compared with the morcellised grafting technique. Bone block augmentation of tendon-implant interfaces results in more reliable functional and histological outcomes, with a return to pre-operative levels of weight-bearing by 24 weeks.

Surgeons treating fractures with many small osteochondral fragments have often expressed the clinical need for an adhesive to join such fragments, as an adjunct to standard implants. If an adhesive would maintain alignment of the articular surfaces and subsequently heal it could result in improved clinical outcomes. However, there are no bone adhesives available for clinical indications and few pre-clinical models to assess safety and efficacy of adhesive biomaterial candidates. A bone adhesive candidate based on water, α-TCP and an amino acid phosphoserine was evaluated in-vivo in a novel murine bone core model (preliminary results presented EORS 2019) in which excised bone cores were glued back in place and harvested @ 0, 3, 7, 14, 28 and 42days. Adhesive pull-out strength was demonstrated 0–28 days, with a dip at 14 days increasing to 11.3N maximum. Histology 0–42 days showed the adhesive progressively remodelling to bone in both cancellous and cortical compartments with no signs of either undesirable inflammation or peripheral ectopic bone formation. These favourable results suggested translation to a large animal model. A porcine dental extraction socket model was subsequently developed where dental implants were affixed only with the adhesive. Biomechanical data was collected @ 1, 14, 28 and 56 days, and histology at 1,14,28 and 56 days. Adhesive strength assessed by implant pull-out force increased out to 28 days and maintained out to 56 days (282N maximum) with failure only occurring at the adhesive bone interface. Histology confirmed the adhesive's biocompatibility and osteoconductive behavior. Additionally, remodelling was demonstrated at the adhesive-bone interface with resorption by osteoclast-like cells and followed by new bone apposition and substitution by bone. Whilst the in-vivo dental implant data is encouraging, a large animal preclinical model is needed (under development) to confirm the adhesive is capable of healing, for example, loaded osteochondral bone fragments. Acknowledgements: The murine study was supported, in part, by the Swedish Foundation for Strategic Research (#RMA15-0110)

Introduction. Femoral head osteonecrosis (FHO) is a condition in which the inadequate blood supply disrupts osteogenic-angiogenic coupling that results in diminishment of femoral perfusion and ends up with FHO. The insufficient knowledge on molecular background and progression pattern of FHO and the restrictions in obtaining human samples bring out the need for a small animal trauma model to research FHO aetiology. Hence, this study aims to develop a mouse trauma model to elucidate the molecular mechanisms behind FHO. Method. Left femoral head was dislocated from the hip joint, ligamentum teres was cut, and a slight circular incision was done around the femoral neck of 8-week-old male C57BL/6J mice to disrupt the blood supply to femoral head. Right hip joint was left unoperated as control. Animals (n=5 per time point) were sacrificed on 2-3-4-6-8-10-12 weeks, and ex-vivo µCT was taken to assess bone structural parameters. Haematoxylin/eosin (HE)- and immunohistochemical-staining (IHCS) for CD31 and EMCN were done to observe histology and marrow-specific H-type vascular structures, respectively. Result. μCT assessment showed trabecular bone loss and decreased BV/TV from 2 to 8 weeks in FHO side. HE staining displayed the increased number of empty lacunae was observed in FHO side as early as 24h after operation. By 4. th. week, IHCS results displayed the invasion of the epiphyseal plate by H-type blood vessels in FHO side, while the epiphyseal plate was observed intact in control side. Also, by 6. th. week the HE-staining showed the presence of bone marrow necrosis and bone fat accumulation in FHO side. Conclusion. Trabecular bone loss, increased number of empty lacunae, bone fat imbalance and bone marrow necrosis are reported as the signs of osteonecrosis. Thus, our results are coherent with the literature and indicated that we were able to effectively generate a trauma model for FHO in mice for the first time in literature

Due to well-known disadvantages of the autologous bone graft, many alternatives have been studied for a reliable spinal fusion. Herein, we aimed to investigate the effects of human recombinant epidermal growth factor (EGF) on posterolateral lumbar fusion in a rat model. 36 male SD rats underwent posterolateral fusion at L4-5 level. They were randomly assigned to 3 groups: Sham control group, Hydoxyapatite β-tricalcium phosphate (HA/β-TCP) group and HA/β-TCP + EGF group. Rats were euthanized at 8 weeks post-surgery. 6 rats from each group were selected for manual palpation examination, micro-computed tomography analysis and histologic analysis; and the rest was used for biomechanical analysis. Based on manual palpation, there was no fusion in the sham control group. Fusion rate was 33.3% in the HA/β-TCP group and 66.7% in the HA/β-TCP + EGF group (p=0.085). Micro-CT results revealed that new bone formation was higher in the HA/β-TCP + EGF group (BV/TV: 40% vs. 65%) (p=0.004). Histologically newly formed bone tissue was more pronounced in the EGF group and compacted and bridging bone spicules were observed. The median maximum bending moment values were 0.51 Nmm (0.42– 0.59), 0.73 Nmm (0.49– 0.88) and 0.91 Nmm (0.66– 1.03) in the sham control, HA/β-TCP and HA/β-TCP + EGF groups, respectively (p=0.013). The median stiffness values were 1.69 N/mm (1.12–2.18), 1.68 N/mm (1.13–2.74) and 3.10 N/mm (1.66–4.40) as in the previous order (p=0.087). This study demonstrates that EGF enhances posterolateral lumbar fusion in the rat model. EGF in combination with ceramic grafts increased the fusion rates.

Translational models for OA have used a variety of small (mouse, rat) and large (sheep, pig) animal models to evaluate the efficacy of a specific therapy. Clinical trials based on the results of these animal models have yielded mixed results with respect to the treatment of the disease. Due to greater stringency in EU regulations in the use of animal models for research, ex vivo models of OA (e.g. cartilage explants, bioreactors) are being developed to mimic human joint motion as well as the inflammatory milieu (e.g. IL-1β) that can be used to understand efficacy of therapy in a physiological environment. The development of these models can enable therapies to undergo clinical trials in patients without the necessity for long-term animal studies. This presentation will describe the state of the art in this field and will discuss whether there is potential to speed up translation from bench to bedside in the future

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro. The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane. Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs. This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment