During the last decades, several research groups have used bisphosphonates for local application to counteract secondary bone resorption after bone grafting, to improve implant fixation or to control bone resorption caused by bone morphogenetic proteins (BMPs). We focused on zoledronate (a bisphosphonate) due to its greater antiresorptive potential over other bisphosphonates. Recently, it has become obvious that the carrier is of importance to modulate the concentration and elution profile of the zoledronic acid locally. Incorporating one fifth of the recommended systemic dose of zoledronate with different apatite matrices and types of bone defects has been shown to enhance bone regeneration significantly in vivo. We expect the local delivery of zoledronate to overcome the limitations and side effects associated with systemic usage; however, we need to know more about the bioavailability and the biological effects. The local use of BMP-2 and zoledronate as a combination has a proven additional effect on bone regeneration. This review focuses primarily on the local use of zoledronate alone, or in combination with bone anabolic factors, in various preclinical models mimicking different orthopaedic conditions. Cite this article: I. Qayoom, D. B. Raina, A. Širka, Š. Tarasevičius, M. Tägil, A. Kumar, L. Lidgren. Anabolic and antiresorptive actions of locally delivered bisphosphonates for bone repair: A review. Bone Joint Res 2018;7:548–560. DOI: 10.1302/2046-3758.710.BJR-2018-0015.R2

Current strategies for bone repair have accepted limitations and the search for synthetic graft materials or for scaffolds that will support ex vivo bone tissue engineering continues. Bioprospecting has led to increased interest in potential applications for marine organisms and their by-products and biomimetic strategies have led to the investigation of naturally occurring porous structures as templates for bone growth. As a rich source of mineralising porous organisms, our seas and oceans could provide new directions for bone tissue engineering that may enhance in vivo and ex vivo bone formation

Abstract. 3D printing of synthetic scaffolds mimicking natural bone chemical composition, structure, and mechanical properties is a promising approach for repairing bone injuries. Direct ink writing (DIW), a type of 3D printing, confers compatibility with a wide range of materials without exposing these materials to extreme heat. Optimizing ink properties such as filament formation capabilities, shear-thinning, and high storage modulus recovery would improve DIW fabrication characteristics. In this study, composite inks based on biodegradable polycaprolactone (PCL), reinforced with nano-hydroxyapatite (HAp), and loaded with vancomycin were designed and evaluated for their rheological properties, wettability, mechanical properties, and antimicrobial properties. The formulated composite inks displayed a shear-thinning behaviour exhibited storage modulus recovery percentages above 80% for all formulations, which is essential for extrusion deposition by DIW at room temperature. Ink formulations were able to form fully interconnected lattice scaffolds with porosities ranging from 42% to 65%. Increasing the HAp concentrations from 55% to 85% w/w increased the shear thinning behaviour and reduced the printed filament width to more closely match the nozzle diameter; this indicates higher HAp proportion reduces ink shrinkage. The scaffold had high wettability at HAp proportions above 65% w/w and the compressive elastic modulus of DIW printed scaffolds exhibited within the range of trabecular bone. Antimicrobial activity was apparent from the agar diffusion assay; zones of inhibition ranging from 15.82 ± 0.25 mm and 20.06 ± 0.25 mm were observed after 24 hr for composite scaffolds loaded with 3% and 9% w/w vancomycin respectively. Vancomycin-loaded PCL/HAp composite inks were developed, displaying good printability, wettability, mechanical properties, and antimicrobial properties, making them an attractive choice for bone repair and regeneration. 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 literature on fracture repair has been reviewed. The traditional concepts of delayed and nonunion have been examined in terms of the phased and balanced anabolic and catabolic responses in bone repair. The role of medical manipulation of these inter-related responses in the fracture healing have been considered

1. A study has been made of the repair of bony defects in the calvaria of albino rats. 2. An accelerated rate of bone repair was observed in experimental defects into which chondroitin sulphate-treated demineralised bone was implanted. 3. Acid-soluble collagen reconstituted with chondroitin sulphate was also more effective as an implant than was acid-soluble collagen reconstituted with sodium chloride. 4. It is concluded from these studies that chondroitin sulphate treatment accelerated the rate of new bone formation induced by demineralised bone, by reconstituted acid-soluble collagen, and to a lesser extent by Gelfoam. It was also found that demineralised bone and fresh homogenous bone promoted bone repair, but that chondroitin sulphate-treated demineralised bone promoted the most rapid rate of bone repair among the substances tested. 5. The possible role of chondroitin sulphate in bone formation is discussed

Tissue regeneration using growth factors has disadvantages while needing to use supraphysiological growth factor concentrations. Gene therapy has been proposed as alternative. Unfortunately, drawbacks such as the use of viruses and the inefficiency of non-viral systems jeopardize clinical translation. mRNA-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. mRNA molecules can be chemically modified in order to improve stability and immunogenicity. Chemically modified mRNA (cmRNA) is much more efficient than pDNA in delivering genes into the cell. The combination of biomaterials with cmRNA is interesting for the tissue engineering and regenerative medicine field. The resulting construct, known as Transcript-Activated Matrix, may act as a cmRNA delivery platform while supporting cell proliferation, extracellular matrix deposition and ultimately de novo tissue formation. Our work and the work of others demonstrated that the use of Transcript-Activated Matrix prolonged transgene expression and enhanced protein translation. This presentation will provide an overview of ongoing research from our group on cmRNA for improving bone repair with a particular focus on Transcript-Activated Matrix for enhancing osteogenesis. Results of our investigation in vitro with stem cells, ex vivo using tissue culture and in vivo using rat models will be presented

Accidents, osteoporosis or cancer can cause severe bone damage requiring grafts to heal. All current grafting methods have disadvantages including scarcity and infection/rejection risks. An alternative is therefore needed. Hydroxyapatite/calcium carbonate (HA/CC) scaffolds mimic the mineral bone composition but lack growth factors present in auto- and allografts, limiting their osteoinductive capacity. We hypothesize that this will increase the osteogenicity and osteoinductivity of scaffolds through the presence of growth factors. The objectives of this study are to develop and mass-produce grafts with enhanced osteoinductive capacity.

HA/CC scaffolds were cultured together with umbilical cord mesenchymal stem cells in bioreactors so that they adhere to the surface and deposit growth factors. Cells growing on the scaffolds are confirmed by Alamar blue assays, SEM, and confocal microscopy. ELISA and IHC are used to assess the growth factor content of the finished product.

It has been confirmed that cells attach to the scaffolds and proliferate over time when grown in bioreactors. Dynamic seeding of cells is clearly advantageous for cell deposits, equalizing the amount of cells on each scaffold granule.

Hydroxyapatite/calcium carbonate scaffolds support cell-growth. This should be confirmed by further research, including Quantification of BMPs and other indicators of osteogenic differentiation such as Runx2, osteocalcin and ALP is pending, and amounts are expected to be increased in enhanced scaffolds and in-vivo implantation.

Failure of bone repair is a challenging problem in the management of fractures. There is a limited supply of autologous bone grafts for treating nonunions, with associated morbidity after harvesting. There is need for a better source of cells for repair. Mesenchymal stem cells (MSCs) hold promise for healing of bone because of their capacity to differentiate into osteoblasts and their availability from a wide variety of sources. Our review aims to evaluate the available clinical evidence and recent progress in strategies which attempt to use autologous and heterologous MSCs in clinical practice, including genetically-modified MSCs and those grown on scaffolds. We have compared various procedures for isolating and expanding a sufficient number of MSCs for use in a clinical setting. There are now a number of clinical studies which have shown that implantation of MSCs is an effective, safe and durable method for aiding the repair and regeneration of bone

Minimally invasive surgery for the restoration of bone tissues lost due to diseases and trauma is preferred by the health care system as the related costs are continuously increasing. Recently, efforts have been paid to optimize injectable calcium phosphate (CaP) cements which have been recognized as excellent alloplastic material for osseous augmentation because of their unique combination of osteoconductivity, biocompatibility and mouldability. The sol-gel synthesis approach appears to be the most suitable route towards performing injectable calcium phosphates. Different strategies used to prepare bioactive and osteoinductive injectable CaP are reported. CaP gels complexed with phosphoserine-tethered poly(ε-lysine) dendrons (G3-K PS) designed to interact with the ceramic phase and able to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) is discussed. Recently, attention has been given to the modification of hydroxyapatite with Strontium (Sr) due to its dual mode of action, simultaneously increasing bone formation (stimulating osteoblast differentiation) while decreasing bone resorption (inhibiting osteoclast differentiation). The effect of systems based on strontium modified hydroxyapatite (Sr-HA) at different composition on proliferation and osteogenic differentiation of hMSC is described. One more approach is based on the use of antimicrobial injectable materials. It has been demonstrated that some imidazolium, pyridinium and quaternary ammonium ionic liquids (IL) have antimicrobial activity against some different clinically significant bacterial and fungal pathogens. Here, we report several systems based on IL at different alkyl-chain length incorporated in Hydroxyapatite (HA) through the sol-gel process to obtain an injectable material with simultaneous opposite responses toward osteoblasts and microbial proliferation.

Material-based strategies seek to engineer synthetic microenvironments that mimic the characteristics of physiological extracellular matrices for applications in regenerative therapies, including bone repair and regeneration. In our group, we identified a specific chemistry, poly(ethyl acrylate) (PEA), able to induce the organization of fibronectin (FN), upon adsorption of the protein, into fibrillar networks similar to the physiological ones, leading to enhanced cellular response, in terms of cell adhesion and differentiation. In this work, we exploit these FN networks to capture and present growth factors (GF) in combination with the integrin binding domain of FN during bone tissue healing. Fibrillar conformation of FN adsorbed on PEA favors the simultaneous availability of the GF binding domain (FNIII12–14) next to the integrin binding region (FNIII9–10), compared to poly(methyl acrylate) (PMA), a material with similar chemistry, where FN adopts a globular conformation. The combined exposure of specific adhesive sequences recognized by integrins and GF binding domains was found to improve the osteogenic differentiation of mesenchymal stem cells. A higher expression of bone proteins was found when BMP2 is bound or sequestered on the material surface versus its administration in the culture media in vitro. The potential of this system as recruiter of GFs was also investigated in a critical-size bone segmental defect in mouse. The synergistic integrin-GF signalling, induced by fibrillar FN, promoted bone formation in vivo with lower BMP2 doses than current technologies. Furthermore, we optimized the system for its potential use in translational research, seeking to address the clinical need of using biocompatible and biodegradable material implants. Polycaprolactone scaffolds were synthesized and coated with a thin layer of plasma- polymerized PEA that recruits and efficiently presents GF during healing of critical size defects. The material-driven FN fibrillogenesis provides a new strategy to efficiently reduce the GF doses administrated in bone regenerative therapies

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

Mesenchymal Stromal Cells (MSC) are promising therapies for fracture healing. However, undifferentiated MSC may act only through an inductive paracrine effect without direct bone formation. Here, we developed an injectable product constituted of human bone-forming cells derived from bone marrow (BM)-MSC (ALLO-P2) that display more potent bone repair properties not only by stimulating host osteoinduction but also by direct bone formation. In vitro, ALLO-P2 overexpressed markers such as ALP compared to BM-MSC isolated from the same donors, suggesting their engagement into the osteogenic lineage. In vivo, a single dose of ALLO-P2 significantly enhanced bone neoformation 14 days post-administration over the calvaria of NMRI-Nude mice compared to the control excipient. Histological analyses and mouse/human type I collagen double-immunolabelling revealed the presence of mineralized bone nodules of mixed host and donor origins in mice administered with ALLO-P2. Together, these results show that ALLO-P2 is a potential promising clinical candidate to promote bone repair, since it can be produced at high yields, is injectable and boosts ossification mechanisms involved in the physiological repair process

During the last decades numerous studies have reported the critical impact of physical activity on bone repair. While most studies have evaluated the tissue response to the local mechanical environment within the fracture gap, there is a lack of information on the systemic role of physical activity during fracture healing. Therefore, the aim of this study was to standardize the mechanical environment in the fracture gap by developing a rotationally and axially stable murine fracture model, and thereby to analyze the systemic influence of physical activity on early bone repair. After stable fixation of a closed femoral fracture, mice (n=18) were housed in cages supplied with running wheels (running distance > 500m/d). At 2 weeks animals were sacrificed and bones were prepared for histomorphometric (n=7), biomechanical (n=7), and protein biochemical analyses (n=4). Additional mice (n=22), which were housed in standard cages, served as controls. Histomorphometric evaluation showed no influence of increased physical activity on bone repair in terms of callus size and tissue composition. Accordingly, also biomechanical testing of the callus revealed no differences between both groups in rotational stiffness, peak rotation angle, and load at failure. Western blot analyses demonstrated no alterations in callus expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) after daily running when compared to controls. We conclude that increased physical activity under standardized mechanical conditions in the fracture gap does not affect early bone repair in mice

The human amniotic membrane (hAM), derived from the placenta, possesses a low (nay inexistant) immunogenicity and exerts an anti-inflammatory, anti-fibrotic, antimicrobial, antiviral and analgesic effect. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with adequate mechanical properties (permeability, stability, elasticity, flexibility, resorbability) preventing the proliferation of fibrous tissue and promoting early neovascularization of the surgical site. Cryopreservation and lyophilization, with sometimes additional decellularization process, are the main preservation methods for hAM storage.

We examined the use of hAM in orthopaedic and maxillofacial bone surgery, specially to shorten the induced membrane technique (Gindraux, 2017). We investigated the cell survival in cryopreserved hAM (Laurent, 2014) and the capacity of intact hAM of in vitro osteodifferentiation (Gualdi, 2019). We explored its in vivo osteogenic potential in an ectopic model (Laurent, 2017) and, with Inserm U1026 BioTis, in a calvarial defect (Fenelon, 2018). Still piloted by U1026, decellularization and/or lyophilization process were developed (Fenelon, 2019) and, processed hAM capacities was assessed for guided bone regeneration (Fenelon 2020) and induced membrane technique (Fenelon, 2021) in mice.

We reported a limited function of hAM for bone defect management. In this light, we recognized medication-related osteonecrosis of the jaw (MRONJ) as appropriate model of disease to evaluate hAM impact on both oral mucosa and bone healing. We treated height compassionate patients (stage II, III) with cryopreserved hAM. A multicentric randomized clinical study (PHRC-I 2020 funding) will be soon conducted in France (regulatory and ethical authorization in progress).

1. The utilisation of radioactive sulphur in vivo has been demonstrated both macroscopically and microscopically during the preosseous stage of bone repair. 2. The labelled mucopolysaccharide complex, chondroitin sulphuric acid, has been studied during the formation of the medullary and periosteal blastemata in the healing of a fracture. 3. The appearance and possible significance of mast cells adjacent to a fracture, and resulting from the stimulus of trauma, are discussed. 4. Cortisone has been seen to affect the formation of the periosteal cartilaginous blastema and subsequent process of endochondral ossification, with liberation of increased amounts of chondroitin sulphuric acid which was calcified rather than ossified

Bone remodelling is mediated through the synchronism of bone resorption (catabolism) by osteoclasts and bone formation (anabolism) by osteoblasts. Imbalances in the bone remodelling cycle represent an underling cause of metabolic bone diseases such as osteoporosis, where bone resorption exceeds formation (1). Current therapeutic strategies to repair osteoporotic bone fractures focus solely in targeting anabolism or supressing catabolism (2). However, these therapeutics do not reverse the structural damage present at the defect site, ultimately leading to impaired fracture healing, making the repair of osteoporotic fractures particularly challenging in orthopaedics. Herein, we focus on investigating a combined versatile pro-anabolic and anti-catabolic effect of Magnesium (Mg²⁺) to modulate bone cell behaviour (3), to develop an engineered biomimetic bio-instructive biomaterial scaffold structurally designed to enhance bone formation while impeding pathological osteoclast resorption activities to facilitate better bone healing and promote repair.

Pre-osteoblasts MC3T3-E1 (OBs) and osteoclasts progenitors RAW 264.7 (OCs) cell lines were cultured in growth media exposed to increasing concentrations of MgCl₂ (0, 0.5, 1, 10, 25 and 50mM) and the optimal concentration to concurrently promote the differentiation of OBs and inhibit the differentiation or funtion of RANKL-induced OCs was assessed. We next used Fluorescence Lifetime Imaging Microscopy to investigate changes in the metabolic pathways during OBs and OCs differentiation when exposed to increasing MgCl₂ concentrations. We developed a range of magnesium-incorporated collagen scaffolds to permit the spatiotemporal release of Mg²⁺ within the established therapeutic window, and to investigate the behaviour of bone cells in a 3D environment.

In our results, we reported an increase in the expression of the bone formation markers osteocalcin and osteopontin for OBs exposed to 10mM MgCl₂, and a significant downregulation of the osteoclast-specific markers TRAP and cathepsin K in RANKL-induced OCs differentiation when exposed to 25mM MgCl₂. Moreover, 25mM MgCl₂ induced changes in the energy metabolism of OCs from a predominantly oxidative phosphorylation towards a more glycolytic pathway suggesting a regulatory effect of Mg²⁺ in the underlying mechanisms of osteoclasts formation and function. The developed porous collagen-magnesium scaffolds significantly reduced the expression of early osteoclastogenic markers RANK and NFkB, and an elevated expression of the osteogenic markers Runx2 and Col1A1 was reported after 7 days.

Our research to date has provided evidences to demonstrate the potential of Mg²⁺ to concurrently enhance osteogenesis while inhibiting osteoclastogenesis in vitro, potentially introducing new targets for developing therapies to repair osteoporotic bone fractures.

Robust repair relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with extracellular matrix proteins to create a natural environment for the tissue- specific cells. Tropoelastin, the precursor of the elastin, is the extracellular matrix protein responsible for elasticity in diverse tissues, including robust blood vessels. We find that tropoelastin contributes a physical role in elasticity and also substantially to the biology of repairing tissue. The emerging model from a range of our in vivo studies is that tropoelastin encodes direct biological effects and has the versatility to promote repair. We have discovered that tropoelastin substantially improves healing by halving the time to repair bone in small animals and large animal preclinical models; tropoelastin elicits this response with early stage neo-angiogenesis, recruitment of endogenous cells with consistently accelerated repair. This potency is marked by the concerted appearance of blood vessels, tissue and phased cellular contributions that work together to accelerate repair

The osteoclastogenesis is regulated by a complex signaling system between the pro-apoptotic factors (Bax-Cyclin E2-Cdk2) and the tumor necrosis factor family (RANKL-RANK-OPG). Extracorporeal Shock Waves Therapy (ESWT) have recently been used in orthopaedic treatments to induce bone repair, but their mechanisms of action are not sufficiently investigated. So we studied the effect of shock-waves on murine osteoblastic cells. Osteoblast cultures were subjected to a single shock-wave with combinations of low energy intensities (0.05mJ/mm2) and 500 number of shocks (impulses), whereas control cells received no treatment. We valued the cell viability quantifying the expressions of Bax and Opg by PCR. We found an immediate negative effect on cell viability, that occurs with an increase of Bax protein expression after 3 hours of treatment. After a longer time lapse a stimulatory effect on cell proliferation, as reflected by the increase of a G(1)-S phase marker, was observed. In fact, in the following 24, 48 and 72 hours after ESW treatment, we found a stronger association of Cyclin E2 and Cdk2, forming active cyclin E-Cdk2 kinase, compared to untreated cells at the same times. We further explored the molecular mechanism for the ESW induction of osteogenesis: by Real Time PCR an enhancement of Runx2 mRNA, evident 48 hours after the treatment, was found. A link between physical ESW and Runx2 activation has been already demonstrated. ESW-induced. O2- production, followed by tyrosine kinase mediated ERK activation and Runx2 activation, resulted in osteogenic cell growth and maturation. Moreover, we analyzed the cytokines RANK-L and OPG osteoblast expression, involved in regulation of osteoclastogenesis. A decrease in RANK-L/OPG ratio was found, perhaps leading to a reduced osteoclastogenesis. The Shock waves have a repair action on bone and it can been explained by the regulation on osteoclastogenesis by the apoptoic pathway of BAX and OPG

Aims: Extracorporeal Shock Waves Therapy (ESWT) has recently been used in orthopaedic treatments to induce bone repair, but their mechanisms of action are not sufficiently investigated. So we studied the effect of shockwaves on murine osteoblastic cells. Methods: Osteoblast cultures were subjected to a single shockwave with combinations of low energy intensities (0,05mJ/mm2) and 500 number of shocks (impulses), whereas control cells received no treatment. Cell division and apoptosis are interconnected and regulated by cyclins, kinases, Bax and Opg. We valued the cell viability quantifying by PCR the expressions of these molecules which regulate cell-cycle. Results: We found an immediate negative effect on cell viability, that occurs with an increase of Bax protein expression after 3 hours of treatment. After a longer time lapse a stimulatory effect on cell proliferation, as reflected by the increase of a G(1)-S phase marker, was observed. In fact, in the following 24, 48 and 72 hours after ESW treatment, we found a stronger association of Cyclin E2 and Cdk2, forming active cyclin E-Cdk2 kinase, compared to untreated cells at the same times. We further explored the molecular mechanism for the ESW induction of osteogenesis: by Real Time PCR an enhancement of Runx2 mRNA, evident 48 hours after the treatment, was found. A link between physical ESW and Runx2 activation has been already demonstrated. ESW-induced O2- production, followed by tyrosine kinase mediated ERK activation and Runx2 activation, resulted in osteogenic cell growth and maturation. Moreover, we analyzed the cytokines RANK-L and OPG osteoblast expression, involved in regulation of osteoclastogenesis. A decrease in RANK-L /OPG ratio was found, perhaps leading to a reduced osteoclastogenesis. Conclusions: The Shock waves have a repair action on bone and it can been explained by the regulation on osteoclastogenesis by the apoptoic pathway of Bax and Opg

The course of secondary fracture healing typically consists of four major phases including inflammation, soft and hard callus formation, and bone remodeling. Callus formation is promoted by mechanical stimulation, yet little is known about the healing tissue response to strain stimuli over shorter timeframes on hourly and daily basis. The aim of this study was to explore the hourly, daily and weekly variations in bone healing progression and to analyze the short-term response of the repair tissue to well-controlled mechanical stimulation.

A system for continuous monitoring of fracture healing was designed for implantation in sheep tibia. The experimental model was adapted from Tufekci et al. 2018 and consisted of 3 mm transverse osteotomy and 30 mm bone defect resulting in an intermediate mobile bone fragment in the tibial shaft. Whereas the distal and proximal parts of the tibia were fixed with external fixator, the mobile fragment was connected to the proximal part via a second, active fixator. A linear actuator embedded in the active fixator moved the mobile fragment axially, thus stimulating mechanically the tissue in the osteotomy gap via well-controlled displacement being independent from the sheep's functional weightbearing. A load sensor was integrated in the active fixation to measure the force acting in the osteotomy gap. During each stimulation cycle the displacement and force magnitudes were recorded to determine in vivo fracture stiffness. Following approval of the local ethics committee, experiments were conducted on four skeletally mature sheep. Starting from the first day after surgery, the daily stimulation protocols consisted of 1000 loading events equally distributed over 12 hours from 9:00 to 21:00 resulting in a single loading event every 44 seconds. No stimulation was performed overnight.

One animal had to be excluded due to inconsistencies in the load sensor data. The onset of tissue stiffening was detected around the eleventh day post-op. However, on a daily basis, the stiffness was not steadily increasing, but instead, an abrupt drop was observed in the beginning of the daily stimulations. Following this initial drop, the stiffness increased until the last stimulation cycle of the day.

The continuous measurements enabled resolving the tissue response to strain stimuli over hours and days. The presented data contributes to the understanding of the influence of patient activity on daily variations in tissue stiffness and can serve to optimize rehabilitation protocols post fractures.