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Long bone fractures typically heal via formation of an external callus, which helps stabilise the bone fragments. Callus composition and morphology influence the mechanical environment, which in turn regulates the progression of healing. Therefore characterising callus development over time is crucial in understanding this mechanobiological regulation. Although bony callus is often assumed to grow towards the fracture from either side, this is not consistent with observations from large animal studies and clinical cases. Therefore, we sought to quantify the morphology of bony callus over time in a large animal model. Sheep tibiae were x-rayed weekly over eight weeks following an osteotomy (n=5), with fixation allowing up to 10% axial displacement under normal weight-bearing. After scaling radiographs by known landmarks and normalising greyscales, bony callus boundaries were defined by manual segmentation. The lateral callus area and coordinates of its centroid were calculated from each image. The external callus initially formed adjacent to the osteotomy site. Over the first four weeks, callus growth from its outer surfaces was characterised by its centre of area moving outwards and away from the osteotomy, on both proximal and distal fragments. Subsequent weeks showed consolidation and resorption from the outer surface of the callus. Our approach allowed bony callus development to be tracked in individuals throughout healing. Contrary to the view that periosteal bone formation originates distant from the fracture, our data showed bony callus adjacent to the defect from early stages, followed by approximately concentric growth. This discrepancy highlights the need for data specific to experimental conditions, and particularly early stages of healing, for evaluating theoretical models of mechanical regulation.

It is well known that mechanical stimulation promotes indirect fracture healing by triggering callus formation. We investigated the short-term response of healing tissue to mechanical stimulation to compare the changes in tissue stiffness during stimulation and resting phases in a preclinical case-series. Four sheep underwent a tibial osteotomy and were instrumented with a custom-made active fixator which applied a mechanical stimulation protocol of 1000 cycles/day, equally distributed over 12 h, followed by 12 h of rest. During each cycle, a surrogate metric for tissue stiffness was measured, enabling a continuous real-time monitoring of the healing progression. A daily stiffness increase during stimulation and an increase during resting were evaluated for each animal. One animal had to be excluded from the evaluation due to technical reasons. For all included animals, the stiffness began to increase within the second week post-op. A characteristic pattern was observed during daily measurements: the stiffness dropped considerably within the first stimulation cycles followed by a steady rise throughout the rest of the stimulation phase. However, for all included animals, the average daily stiffness increase within the first three weeks post operation was larger during resting than during stimulation (Sheep I: 16.9% vs. −5.7%, Sheep II: 14.7% vs. −1.8%, Sheep III: 8.9% vs. 1.6%). A continuous measurement of tissue stiffness together with a controlled fracture stimulation enabled the investigation of the short-term effects of specific stimulatory parameters, such as resting periods. Resting was identified as a potentially determining factor for bone healing progression. Optimizing the ratio between stimulation and resting may contribute to more robust fracture healing in the future.

Background : Fracture fixation has advanced significantly with the introduction of locked plating and minimally invasive surgical techniques. However, healing complications occur in up to 10% of cases, of which a significant portion may be attributed to unfavorable mechanical conditions at the fracture. Moreover, state-of-the-art plates are prone to failure from excessive loading or fatigue. A novel biphasic plating concept has been developed to create reliable mechanical conditions for timely bone healing and simultaneously improve implant strength. This paper introduces the novel fixation concept and presents preclinical results from a large animal study. Methods : Twenty-four sheep underwent a mid-diaphyseal osteotomy stabilized with either the novel biphasic plate fixator or a control locking plate. Different fracture patterns regarding orientation and localization were investigated. Animals were free to fully bear weight during the post-operative period. After 12 weeks, the healing fractures were evaluated for bone formation using micro-computer tomography and strength and stiffness using biomechanical testing. Additionally, histological evaluation of soft tissue samples with respect to metal wear debris was performed. Results : No plate deformation or failures were observed under full weight bearing with the biphasic plate. Defects stabilized with the biphasic plate demonstrated robust callus formation compared to control group. Torsion tests after plate removal revealed no statistical difference in peak torsion to failure and stiffness for the different fracture patterns stabilized with the biphasic plate. However, the biphasic plate group specimens were 45% stronger (p=0.002) and 48% stiffer (p=0.007) than the controls. No histological signs of metal wear due to the biphasic feature could be found. Conclusions : The biphasic plate concept is aimed at improving the biomechanics of locked plating. The results of this large animal study demonstrate the feasibility and clinical potential of this novel stabilization concept.

Adaptive finite element models have allowed researchers to test hypothetical relationships between the local mechanical environment and the healing of bone fractures. However, their predictive power has not yet been demonstrated by testing hypotheses ahead of experimental testing. In this study, an established mechano-biological scheme was used in an iterative finite element simulation of sheep tibial osteotomy healing under a hypothetical fixation regime, “inverse dynamisation”. Tissue distributions, interfragmentary movement and stiffness across the fracture site were compared between stiff and flexible fixation conditions and scenarios in which fixation stiffness was increased at a discrete time-point. The modelling work was conducted blind to the experimental study to be published subsequently. The simulations predicted the fastest and most direct healing under constant stiff fixation, and the slowest healing under flexible fixation. Although low fixation stiffness promoted more callus formation prior to bridging, this conferred little additional stiffness to the fracture in the first 5 weeks. Thus, while switching to stiffer fixation facilitated rapid subsequent bridging of the fracture, no advantage of inverse dynamisation could be demonstrated. In vivo data remains necessary to conclusively test this treatment protocol and this will, in turn, provide an evaluation of the model’s performance. The publication of both hypotheses and their computational simulation, prior to experimental testing, offers an appealing means to test the predictive power of mechano-biological models.

Internal fixation by plate osteosynthesis is the gold standard treatment for distal femur fractures. Despite improvements that preserve the biological conditions for bone healing, there are concerns standard locked plating constructs may be overly stiff. Biphasic plating is a novel concept designed to provide suitable fracture motion and increased implant strength to support early full weight-bearing. This study aims to demonstrate that the Biphasic Plate can be incorporated into a pre-contoured distal femur plate while providing adequate flexibility and increased implant strength. The mechanical performance of the Biphasic Plate (BP) was investigated in comparison to a standard locking plate for the distal femur (LCP-DF). Constructs were formed by mounting the implants on a bone substitute. The construct stiffness and strength under axial loading and the magnitude of interfragmentary movement were determined using finite element analysis. The Biphasic Plate exhibited a bi-linear stiffness response; at low loads, the BP construct was 55% more compliant and at high loads 476% stiffer than the LCP-DF. The Biphasic Plate provided more consistent interfragmentary movement over a wider loading range. At partial weight-bearing loads, the Biphasic Plate produced larger interfragmentary movements (0.18 vs. 0.04 mm). However, at loads equivalent to full weight-bearing, the maximum movements were substantially smaller than the LCP-DF construct (1.5 vs. 3.5 mm). The increased flexibility at low loads was provided without sacrificing implant strength with peak stress in the Biphasic Plate 63% lower than the LCP-DF construct. The biphasic plating concept can be successfully incorporated into anatomically contoured distal femur plates while providing adequate flexibility and increasing implant strength.

Iterative computational models have been used to investigate the regulation of bone fracture healing by local mechanical conditions. Although their predictions replicate some mechanical responses and histological features, they do not typically reproduce the predominantly radial hard callus growth pattern observed in larger mammals. We hypothesised that this discrepancy results from an artefact of the models' initial geometry. Using axisymmetric finite element models, we demonstrated that pre-defining a field of soft tissue in which callus may develop introduces high deviatoric strains in the periosteal region adjacent to the fracture. These bone-inhibiting strains are not present when the initial soft tissue is confined to a thin periosteal layer. As observed in previous healing models, tissue differentiation algorithms regulated by deviatoric strain predicted hard callus forming remotely and growing towards the fracture. While dilatational strain regulation allowed early bone formation closer to the fracture, hard callus still formed initially over a broad area, rather than expanding over time. Modelling callus growth from a thin periosteal layer successfully predicted the initiation of hard callus growth close to the fracture site. However, these models were still susceptible to elevated deviatoric strains in the soft tissues at the edge of the hard callus. Our study highlights the importance of the initial soft tissue geometry used for finite element models of fracture healing. If this cannot be defined accurately, alternative mechanisms for the prediction of early callus development should be investigated.

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL-TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL-TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

Die Behandlung segmentaler Knochendefekte ist vorwiegend auf die Transplantation von autologem oder allogenem Knochen limitiert. Diese Transplantate besitzen zwar osteokonduktive oder -induktive Eigenschaften, sind jedoch auch mit entscheidenden Nachteilen wie Entnahmemorbiditat, begrenzter Verfugbarkeit und unvollstandiger Transplantatintegration behaftet. Das Ziel der vorliegenden Studie war es daher, ein therapeutisches Alternativkonzept zu entwickeln. Das regenerative Potenzial von 2 grundsatzlich verschiedenen Gerusttragern (Scaffolds), der eine bestehend aus e-Polycaprolacton-β-Trikalziumphosphat (mPCL-TCP) und der andere aus Seide und Hydroxylapatit (Seide-HA) wurde mit der Knochenneubildung nach Transplantation autologer Spongiosa (ABG) verglichen. Defekte kritischer Grose in der Schaftibia blieben unbehandelt, wurden mit autologer Spongiosa vom Beckenkamm, mit einem mPCL-TCP oder mit Seide-HA-Scaffolds rekonstruiert (n = 6). Die Tiere wurden fur 3 Monate beobachtet, sie durften voll belasten. Es wurden Rontgenbilder sowie computertomographische Aufnahmen angefertigt und die Aufnahmen quantitativ ausgewertet. Zur biomechanischen Testung wurde ein Torsionstest mit einer Geschwindigkeit von 0,5°/s durchgefuhrt. In den Seide-HA-Scaffolds wurde eine nur geringe Knochenneubildung observiert, mehr Knochen bildete sich in den mPCL-TCP-Scaffolds, der die Defekte partiell uberbruckte. Eine komplette Uberbruckung aller Defekte wurde nach Rekonstruktion mit ABG beobachtet. Es wurde eine deutliche Varianz innerhalb der Gruppen festgestellt. Die biomechanische Testung zeigte hohere Werte fur die ABG- und mPCL-TCP-Gruppe im Vergleich zur Seide-HA-Gruppe. Ein signifikanter Unterschied zwischen ABG und Seide-HA konnte jedoch nicht zwischen ABG und mPCL-TCP bzw. mPCL-TCP und Seide-HA festgestellt werden. Die Ergebnisse zeigen, dass mPCL-TCP-Scaffolds eine Alternative zur Applikation von autologem Knochen darstellen konnen. Die Kombination mit osteogenen Zellen, Stimulanzien und Wachstumsfaktoren bietet hierbei eine attraktive Moglichkeit, um eine weitere Steigerung der Knochenneubildung zu bewirken.

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Secondary fracture healing in long bones leads to the successive formation of intricate patterns of tissues in the newly formed callus. The main aim of this work was to quantitatively describe the topology of these tissue patterns at different stages of the healing process and to generate averaged images of tissue distribution. This averaging procedure was based on stained histological sections (2, 3, 6, and 9 weeks post-operatively) of 64 sheep with a 3 mm tibial mid-shaft osteotomy, stabilized either with a rigid or a semi-rigid external fixator. Before averaging, histological images were sorted for topology according to six identified tissue patterns. The averaged images were obtained for both fixation types and the lateral and medial side separately. For each case, the result of the averaging procedure was a collection of six images characterizing quantitatively the progression of the healing process. In addition, quantified descriptions of the newly formed cartilage and the bone area fractions (BA/TA) of the bony callus are presented. For all cases, a linear increase in the BA/TA of the bony callus was observed. The slope was greatest in the case of the most rigid stabilization and lowest in the case of the least stiff. This topological description of the progression of bone healing will allow quantitative validation (or falsification) of current mechano-biological theories.

Currently, well-established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, however they are limited in access and availability and associated with donor site morbidity, haemorrhage, risk of infection, insufficient transplant integration, graft devitalisation, and subsequent resorption resulting in decreased mechanical stability. As a result, recent research focuses on the development of alternative therapeutic concepts. The field of tissue engineering has emerged as an important approach to bone regeneration. However, bench to bedside translations are still infrequent as the process towards approval by regulatory bodies is protracted and costly, requiring both comprehensive in vitro and in vivo studies. The subsequent gap between research and clinical translation, hence commercialization, is referred to as the ‘Valley of Death’ and describes a large number of projects and/or ventures that are ceased due to a lack of funding during the transition from product/technology development to regulatory approval and subsequently commercialization. One of the greatest difficulties in bridging the Valley of Death is to develop good manufacturing processes (GMP) and scalable designs and to apply these in pre-clinical studies. In this article, we describe part of the rationale and road map of how our multidisciplinary research team has approached the first steps to translate orthopaedic bone engineering from bench to bedside byestablishing a pre-clinical ovine critical-sized tibial segmental bone defect model and discuss our preliminary data relating to this decisive step.

BackgroundIn osteochondral defects, subchondral bone, as a load-bearing structure, is believed to be important for bone and cartilage regeneration.HypothesisA stiff scaffold creates better conditions for bone formation and cartilage regeneration than does a softer one.Study DesignControlled laboratory study.MethodsCritical osteochondral defects were created in the femoral condyles of 24 sheep. Subchondral bone was reconstructed with a stiff scaffold or a modified softer one, with untreated defects serving as controls. The repair response was evaluated with mechanical, histological, and histomorphometrical techniques at 3 and 6 months postoperatively.ResultsThe elastic modulus of regenerated fibrocartilage over the stiff scaffold tended to be higher than in the soft scaffold group (61 % vs 46% of healthy cartilage) at 3 months. No difference was determined at 6 months; all were well below healthy cartilage. Treated defects showed substantial degradation of the soft scaffold with surrounding sclerotic bone at 3 and 6 months. In contrast, degradation of the stiff scaffold was slower and occurred together with continuous osseous replacement.ConclusionStiff scaffolds were found to improve bone regeneration. In contrast, soft scaffolds provided less support, and consequently subchondral bone became sclerotic. Although regenerated cartilage formed over the stiff scaffolds at 3 months, and these exhibited better mechanical properties than did the soft scaffold group, the mechanical properties in both treated groups were the same at 6 months, not dissimilar to that of tissue formed in the untreated specimens and inferior to native articular cartilage.Clinical RelevanceThe results imply that subchondral defect filling in clinical settings advances bone regeneration and should have a comparable stiffness to that of healthy subchondral bone rather than being too flexible. Degradation of resorbable materials and consequently the loss of stiffness may compromise the healing of critical defects.

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Background: Bone-healing is known to be sensitive to the mechanical stability of fixation. However, the influence on healing of the individual components of fixation stiffness remains unclear. The aim of this study was to investigate the relationship between the initial in vitro fixation stiffness and the strength and stiffness of the callus after nine weeks. We hypothesized that axial stiffness would determine the healing outcome. Methods: A standardized midshaft osteotomy of the right tibia was performed on Merino-mix sheep and was stabilized with either one of four monolateral external fixators or one of two tibial nails inserted without reaming. The in vitro stiffness of fixation was determined in six loading conditions (axial compression, torsion, as well as bending and shear in the anteroposterior and mediolateral planes) on ovine tibial specimens. Stiffness was calculated by relating displacements of the fracture fragments, determined by means of attached optical markers, and the loads applied by a materials testing machine. Torsional testing until failure of the explanted tibiae was performed with use of a standard materials testing machine after nine weeks of healing to determine the failure moment and the torsional stiffness of the healed tibia. Results: External fixation in sheep generally resulted in higher fixation stiffness than did conventional unreamed tibial nailing. The use of angle-stable locking screws in tibial nailing resulted in fixation stiffness comparable with that of external fixation. The highest torsional moment to failure was observed for the external fixator with moderate axial stiffness and high shear stiffness. The fixator with the highest axial stability did not result in the highest failure moment. Low axial stability in combination with low shear stability resulted in the lowest failure moment. Conclusions: In this study, a clear relationship between the stability of fixation and the mechanical strength of the healing tibia was seen. Moderate levels of axial stability were associated with the highest callus strength and stiffness. Clinical Relevance: Optimizing axial stability and limiting shear instability appear to be important for creating conditions for timely fracture-healing.

The treatment of large segmental bone defects remains a significant clinical challenge. Due to limitations surrounding the use of bone grafts, tissue-engineered constructs for the repair of large bone defects could offer an alternative. Before translation of any newly developed tissue engineering (TE) approach to the clinic, efficacy of the treatment must be shown in a validated preclinical large animal model. Currently, biomechanical testing, histology, and microcomputed tomography are performed to assess the quality and quantity of the regenerated bone. However, in vivo monitoring of the progression of healing is seldom performed, which could reveal important information regarding time to restoration of mechanical function and acceleration of regeneration. Furthermore, since the mechanical environment is known to influence bone regeneration, and limb loading of the animals can poorly be controlled, characterizing activity and load history could provide the ability to explain variability in the acquired data sets and potentially outliers based on abnormal loading. Many approaches have been devised to monitor the progression of healing and characterize the mechanical environment in fracture healing studies. In this article, we review previous methods and share results of recent work of our group toward developing and implementing a comprehensive biomechanical monitoring system to study bone regeneration in preclinical TE studies.

Background: Bone healing is sensitive to the initial mechanical conditions with tissue differentiation being determined within days of trauma. Whilst axial compression is regarded as stimulatory, the role of interfragmentary shear is controversial. The purpose of this study was to determine how the initial mechanical conditions produced by interfragmentary shear and torsion differ from those produced by axial compressive movements. ----- ----- Methods: The finite element method was used to estimate the strain, pressure and fluid flow in the early callus tissue produced by the different modes of interfragmentary movement found in vivo. Additionally, tissue formation was predicted according to three principally different mechanobiological theories. ----- ----- Findings: Large interfragmentary shear movements produced comparable strains and less fluid flow and pressure than moderate axial interfragmentary movements. Additionally, combined axial and shear movements did not result in overall increases in the strains and the strain magnitudes were similar to those produced by axial movements alone. Only when axial movements where applied did the non-distortional component of the pressure–deformation theory influence the initial tissue predictions. ----- ----- Interpretation: This study found that the mechanical stimuli generated by interfragmentary shear and torsion differed from those produced by axial interfragmentary movements. The initial tissue formation as predicted by the mechanobiological theories was dominated by the deformation stimulus.

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

Successful healing of long bone fractures is dependent on the mechanical environment created within the fracture, which in turn is dependent on the fixation strategy. Recent literature reports have suggested that locked plating devices are too stiff to reliably promote healing. However, in vitro testing of these devices has been inconsistent in both method of constraint and reported outcomes, making comparisons between studies and the assessment of construct stiffness problematic. Each of the methods previously used in the literature were assessed for their effect on the bending of the sample and concordant stiffness. The choice of outcome measures used in in vitro fracture studies was also assessed. Mechanical testing was conducted on seven hole locked plated constructs in each method for comparison. Based on the assessment of each method the use of spherical bearings, ball joints or similar is suggested at both ends of the sample. The use of near and far cortex movement was found to be more comprehensive and more accurate than traditional centrally calculated inter fragmentary movement values; stiffness was found to be highly susceptible to the accuracy of deformation measurements and constraint method, and should only be used as a within study comparison method. The reported stiffness values of locked plate constructs from in vitro mechanical testing is highly susceptible to testing constraints and output measures, with many standard techniques overestimating the stiffness of the construct. This raises the need for further investigation into the actual mechanical behaviour within the fracture gap of these devices.

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Finite element models of bones can be created by deriving geometry from an X-ray CT scan. Material properties such as the elastic modulus can then be applied using either a single or set of homogeneous values, or individual elements can have local values mapped onto them. Values for the elastic modulus can be derived from the CT density values using an elasticity versus density relationship. Many elasticity–density relationships have been reported in the literature for human bone. However, while ovine in vivo models are common in orthopaedic research, no work has been done to date on creating FE models of ovine bones. To create these models and apply relevant material properties, an ovine elasticity–density relationship needs to be determined. Using fresh frozen ovine tibias the apparent density of regions of interest was determined from a clinical CT scan. The bones were the sectioned into cuboid samples of cortical bone from the regions of interest. Ultrasound was used to determine the elastic modulus in each of three directions – longitudinally, radially and tangentially. Samples then underwent traditional compression testing in each direction. The relationships between apparent density and both ultrasound, and compression modulus in each direction were determined. Ultrasound testing was found to be a highly repeatable non-destructive method of calculating the elastic modulus, particularly suited to samples of this size. The elasticity–density relationships determined in the longitudinal direction were very similar between the compression and ultrasound data over the density range examined. A clear difference was seen in the elastic modulus between the longitudinal and transverse directions of the bone samples, and a transverse elasticity–density relationship is also reported.

The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow-derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep - a model closely resembling human bone formation and structure - were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Bone loss may result from remodelling initiated by implant stress protection. Quantifying remodelling requires bone density distributions which can be obtained from computed tomography scans. Pre-operative scans of large animals however are rarely possible. This study aimed to determine if the contra-lateral bone is a suitable control for the purpose of quantifying bone remodelling. CT scans of 8 pairs of ovine tibia were used to determine the likeness of left and right bones. The deviation between the outer surfaces of the bone pairs was used to quantify geometric similarity. The density differences were determined by dividing the bones into discrete volumes along the shaft of the tibia. Density differences were also determined for fractured and contra-lateral bone pairs to determine the magnitude of implant related remodelling. Left and right ovine tibiae were found to have a high degree of similarity with differences of less than 1.0 mm in the outer surface deviation and density difference of less than 5% in over 90% of the shaft region. The density differences (10–40%) as a result of implant related bone remodelling were greater than left-right differences. Therefore, for the purpose of quantifying bone remodelling in sheep, the contra-lateral tibia may be considered an alternative to a pre-operative control.

Generation of subject-specific finite element (FE) models from computed tomography (CT) datasets is of significance for application of the FE analysis to bone structures. A great challenge that remains is the automatic assignment of bone material properties from CT Hounsfield Units into finite element models. This paper proposes a new assignment approach, in which material properties are directly assigned to each integration point. Instead of modifying the dataset of FE models, the proposed approach divides the assignment procedure into two steps: generating the data file of the image intensity of a bone in a MATLAB program and reading the file into ABAQUS via user subroutines. Its accuracy has been validated by assigning the density of a bone phantom into a FE model. The proposed approach has been applied to the FE model of a sheep tibia and its applicability tested on a variety of element types. The proposed assignment approach is simple and illustrative. It can be easily modified to fit users' situations.

To analyse mechanotransduction resulting from tensile loading under defined conditions, various devices for in vitro cell stimulation have been developed. This work aimed to determine the strain distribution on the membrane of a commercially available device and its consistency with rising cycle numbers, as well as the amount of strain transferred to adherent cells. The strains and their behaviour within the stimulation device were determined using digital image correlation (DIC). The strain transferred to cells was measured on eGFP-transfected bone marrow-derived cells imaged with a fluorescence microscope. The analysis was performed by determining the coordinates of prominent positions on the cells, calculating vectors between the coordinates and their length changes with increasing applied tensile strain. The stimulation device was found to apply homogeneous (mean of standard deviations approx. 2% of mean strain) and reproducible strains in the central well area. However, on average, only half of the applied strain was transferred to the bone marrow-derived cells. Furthermore, the strain measured within the device increased significantly with an increasing number of cycles while the membrane's Young's modulus decreased, indicating permanent changes in the material during extended use. Thus, strain magnitudes do not match the system readout and results require careful interpretation, especially at high cycle numbers.

After bone fracture, various cellular activities lead to the formation of different tissue types, which form the basis for the process of secondary bone healing. Although these tissues have been quantified by histology, their material properties are not well understood. Thus, the aim of this study is to correlate the spatial and temporal variations in the mineral content and the nanoindentation modulus of the callus formed via intramembranous ossification over the course of bone healing. Midshaft tibial samples from a sheep osteotomy model at time points of 2, 3, 6 and 9 weeks were employed. PMMA embedded blocks were used for quantitative back scattered electron imaging and nanoindentation of the newly formed periosteal callus near the cortex. The resulting indentation modulus maps show the heterogeneity in the modulus in the selected regions of the callus. The indentation modulus of the embedded callus is about 6 GPa at the early stage. At later stages of mineralization, the average indentation modulus reaches 14 GPa. There is a slight decrease in average indentation modulus in regions distant to the cortex, probably due to remodelling of the peripheral callus. The spatial and temporal distribution of mineral content in the callus tissue also illustrates the ongoing remodelling process observed from histological analysis. Most interestingly the average indentation modulus, even at 9 weeks, remains as low as 13 GPa, which is roughly 60% of that for cortical sheep bone. The decreased indentation modulus in the callus compared to cortex is due to the lower average mineral content and may be perhaps also due to the properties of the organic matrix which might be different from normal bone.

A considerable number of international research groups as well as commercial entities work on the development of new bone grafting materials, carriers, growth factors and specifically tissue-engineered constructs for bone regeneration. They are strongly interested in evaluating their concepts in highly reproducible large segmental defects in preclinical and large animal models. To allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools and act as a base for further directions to orthopaedic and tissue engineering developments, specifically translation into the clinic. In this leading opinion paper, we aim to review and critically discuss the different large animal bone defect models reported in the literature. We conclude that most publications provide only rudimentary information on how to establish relevant preclinical segmental bone defects in large animals. Hence, we express our opinion on methodologies to establish preclinical critically sized, segmental bone defect models used in past research with reference to surgical techniques, fixation methods and postoperative management focusing on tibial fracture and segmental defect models.

BACKGROUND: Treatment of proximal humerus fractures in elderly patients is challenging because of reduced bone quality. We determined the in vitro characteristics of a new implant developed to target the remaining bone stock, and compared it with an implant in clinical use. METHODS: Following osteotomy, left and right humeral pairs from cadavers were treated with either the Button-Fix or the Humerusblock fixation system. Implant stiffness was determined for three clinically relevant cases of load: axial compression, torsion, and varus bending. In addition, a cyclic varus-bending test was performed. RESULTS: We found higher stiffness values for the humeri treated with the ButtonFix system--with almost a doubling of the compression, torsion, and bending stiffness values. Under dynamic loading, the ButtonFix system had superior stiffness and less K-wire migration compared to the Humerusblock system. INTERPRETATION: When compared to the Humerusblock design, the ButtonFix system showed superior biomechanical properties, both static and dynamic. It offers a minimally invasive alternative for the treatment of proximal humerus fractures.

Background Grafting of autologous hyaline cartilage and bone for articular cartilage repair is a well-accepted technique. Although encouraging midterm clinical results have been reported, no information on the mechanical competence of the transplanted joint surface is available. Hypothesis The mechanical competence of osteochondral autografts is maintained after transplantation. Study Design Controlled laboratory study. Methods Osteochondral defects were filled with autografts (7.45 mm in diameter) in one femoral condyle in 12 mature sheep. The ipsilateral femoral condyle served as the donor site, and the resulting defect (8.3 mm in diameter) was left empty. The repair response was examined after 3 and 6 months with mechanical and histologic assessment and histomorphometric techniques. Results Good surface congruity and plug placement was achieved. The Young modulus of the grafted cartilage significantly dropped to 57.5% of healthy tissue after 3 months (P < .05) but then recovered to 82.2% after 6 months. The aggregate and dynamic moduli behaved similarly. The graft edges showed fibrillation and, in some cases (4 of 6), hypercellularity and chondrocyte clustering. Subchondral bone sclerosis was observed in 8 of 12 cases, and the amount of mineralized bone in the graft area increased from 40% to 61%. Conclusions The mechanical quality of transplanted cartilage varies considerably over a short period of time, potentially reflecting both degenerative and regenerative processes, while histologically signs of both cartilage and bone degeneration occur. Clinical Relevance Both the mechanically degenerative and restorative processes illustrate the complex progression of regeneration after osteochondral transplantation. The histologic evidence raises doubts as to the long-term durability of the osteochondral repair.

BACKGROUND: Trochlear dysplasia is suspected to have a genetic basis and causes recurrent patellar instability due to insufficient anatomical geometry. Numerous studies about trochlear morphology and the optimal surgical treatment have been carried out, but no attention has been paid to the corresponding patellar morphology.----- ----- PURPOSE: The aim of this study was the evaluation of the patellar morphology in normal and trochlear dysplastic knees. ----- ----- STUDY DESIGN: Biometric analysis. ----- ----- METHODS: Twenty two patellae with underlying trochlear dysplasia (study group--SG) were compared with 22 matched knees with normal trochlear shape (control group--CG) on transverse and sagittal MRI slices. We compared transverse diameter, cartilaginous thickness, Wiberg-index and -angle, length and radius of lateral and medial facet, patellar shape and angle, retropatellar length, and type of trochlear dysplasia. For statistical analysis we used the Wilcoxon signed ranks test. ----- ----- RESULTS: The transverse and sagittal diameter, mean length of medial patellar facet, and mean cartilaginous and subchondral Wiberg-index showed statistical differences between the two groups. ----- ----- CONCLUSIONS: Although the insufficient trochlear depth and decreased lateral trochlear slope are responsible for patellofemoral instability, the patella shows morphological changes in trochlear dysplastic knees. Its overall size and the medial facet are smaller. Although the femoral sulcus angle is larger, the Wiberg-angle and -index are equal to the control group. This may indicate that the patellar morphology may not be a result of missing medial patellofemoral pressure in trochlear dysplastic knees, but a decreased medial patellofemoral traction. This seems to be caused by hypotrophic medial patellofemoral restraints in combination with an increased lateral patellar tilt, both resulting in a decreased tension onto the medial patella facet. Whether there is a genetic component to the patellar morphology remains open.

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

Fracture healing is influenced by fixation stability and experimental evidence suggests that the initial mechanical conditions may determine the healing outcome. We hypothesised that mechanical conditions influence not only the healing outcome, but also the early phase of fracture healing. Additionally, it was hypothesised that decreased fixation stability characterised by an increased shear interfragmentary movement results in a delay in healing. Sixty-four sheep underwent a mid-shaft tibial osteotomy which was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture callus was analysed using radiological, biomechanical and histological techniques. The tibia treated with semi-rigid fixation showed inferior callus stiffness and quality after 6 weeks. At 9 weeks, the calluses were no longer distinguishable in their mechanical competence. The calluses at 9 weeks produced under rigid fixation were smaller and consisted of a reduced fibrous tissue component. These results demonstrate that the callus formation over the course of healing differed both morphologically and in the rate of development. In this study, we provide evidence that the course of healing is influenced by the initial fixation stability. The semi-rigid fixator did not result in delayed healing, but a less optimal healing path was taken. An upper limit of stability required for successful healing remains unknown, however a limit by which healing is less optimal has been determined.

Osteoclasts are specialised bone-resorbing cells. This particular ability makes osteoclasts irreplaceable for the continual physiological process of bone remodelling as well as for the repair process during bone healing. Whereas the effects of systemic diseases on osteoclasts have been described by many authors, the spatial and temporal distribution of osteoclasts during bone healing seems to be unclear so far. In the present study, healing of a tibial osteotomy under standardised external fixation was examined after 2, 3, 6 and 9 weeks (n = 8) in sheep. The osteoclastic number was counted, the area of mineralised bone tissue was measured histomorphometrically and density of osteoclasts per square millimetre mineralised tissue was calculated. The osteoclastic density in the endosteal region increased, whereas the density in the periosteal region remained relatively constant. The density of osteoclasts within the cortical bone increased slightly over the first 6 weeks, however, there was a more rapid increase between the sixth and ninth weeks. The findings of this study imply that remodelling and resorption take place already in the very early phase of bone healing. The most frequent remodelling process can be found in the periosteal callus, emphasising its role as the main stabiliser. The endosteal space undergoes resorption in order to recanalise the medullary cavity, a process also started in the very early phase of healing at a low level and increasing significantly during healing. The cortical bone adapts in its outward appearance to the surrounding callus structure. This paradoxic loosening is caused by the continually increasing number and density of osteoclasts in the cortical bone ends. This study clearly emphasises the osteoclastic role especially during early bone healing. These cells do not simply resorb bone but participate in a fine adjusted system with the bone-producing osteoblasts in order to maintain and improve the structural strength of bone tissue.

Study Design. A numerical analysis of stress shielding of the bone graft in box and cylinder interbody fusion cages was performed. Objectives. To evaluate the stress shielding characteristics of box and cylinder interbody fusion cages for the cervical spine with regard to their rigidity and contiguous pore size. Summary of Background Data. Cage design has been shown to influence loading of the augmented bone graft tissue. In addition, a large contiguous pore design is believed to be important to avoid stress shielding effects. Methods. A two-dimensional axisymmetric, biphasic finite-element model of the cage incorporating the bone graft and the adjacent vertebral bodies was developed. Analysis was performed in two parts. First, the vertebrae were loaded by an axial compressive force, and second, the effect of vertebral penetration by the interbody cage was simulated. Results. Straining of bone graft in the box cage was generally lower than that of the cylinder cage. The strains in the cylinder cage were seen to be more uniformly distributed, whereas in the box cage straining was concentrated in the graft under the endplates. Vertebral penetration by the cylinder cage resulted in significant straining of the bone graft (28% strain), whereas lower strains were determined in the box cage (a maximum of 17% strain). Conclusions. The central pore in the box design does not seem as effective as the fully open cylinder cage in transferring loads to the augmented graft tissue. Early penetration of the adjacent vertebrae by the cylinder cage may provide early postoperative stability and load the graft tissue, thereby imparting the necessary signals for fusion.