The absence of immediate stimulation delays bone healing.

AIM: Secondary bone healing requires an adequate level of mechanical stimulation expressed by the extent of interfragmentary motion in the fracture. However, there is no consensus about when the mechanical stimulation should be initiated to ensure a timely healing response. Therefore, this study aims to compare the effect of the immediate and delayed application of mechanical stimulation in a large animal model. METHODS: Twelve Swiss White Alpine sheep underwent partial osteotomy of a tibia that was stabilised with an active fixator inducing well-controlled mechanical stimulation. Animals were randomly assigned into two groups with different stimulation protocols. The immediate group received daily stimulation (1000 cycles/day) from the first day post-operation, while in the delayed group, stimulation began only on the 22nd day post-operation. Healing progression was evaluated daily by measuring the in vivo stiffness of the repair tissue and by quantifying callus area on weekly radiographs. All animals were euthanised five weeks post-op. Post-mortem callus volume was determined from high-resolution computer tomography (HRCT). RESULTS: Fracture stiffness (p < 0.05) and callus area (p < 0.01) were significantly larger for the immediate group compared to the delayed stimulation group. In addition, the callus volume measured on the post-mortem HRCT showed 319 % greater callus volume for the immediate stimulation group (p < 0.01). CONCLUSIONS: This study demonstrates that a delay in the onset of mechanical stimulation retards fracture callus development and that mechanical stimulation already applied in the early post-op phase promotes bone healing.

Statistical Morphology and Fragment Mapping of Complex Proximal Humeral Fractures.

Background and Objectives: Proximal humerus fractures (PHFs) are common in the elderly, but the treatment results are often poor. A clear understanding of fracture morphology and distribution of cortical bone loss is important for improved surgical decision making, operative considerations, and new implant designs. The aim of this study was to develop a 3D segmentation fracture mapping technique to create a statistical description of the spatial pattern and cortical bone loss of complex PHFs. Materials and Methods: Fifty clinical computed tomography (CT) scans of complex PHFs and their contralateral intact shoulders were collected. In-house software was developed for semi-automated segmentation and fracture line detection and was combined with manual fracture reduction to the contralateral template in a commercial software. A statistical mean model of these cases was built and used to describe probability maps of the fracture lines and cortical fragments. Results: The fracture lines predominantly passed through the surgical neck and between the tuberosities and tendon insertions. The superior aspects of the tuberosities were constant fragments where comminution was less likely. Some fracture lines passed through the bicipital sulcus, but predominantly at its edges and curving around the tuberosities proximally and distally. Conclusions: A comprehensive and systematic approach was developed for processing clinical CT images of complex fractures into fracture morphology and fragment probability maps and applied on PHFs. This information creates an important basis for better understanding of fracture morphology that could be utilized in future studies for surgical training and implant design.

Surgical technique and comparison of autologous cancellous bone grafts from various donor sites in rats.

Autologous cancellous bone graft is the gold standard in large bone defect repair. However, studies using autologous bone grafting in rats are rare. To determine the feasibility of autologous cancellous bone graft harvest from different anatomical donor sites (humerus, ilium, femur, tibia, and tail vertebrae) in rats and compare their suitability as donor sites, a total of 13 freshly euthanized rats were used to describe the surgical technique, determine the cancellous bone volume and microstructure, and compare the cancellous bone collected quantitatively and qualitatively. It was feasible to harvest cancellous bone grafts from all five anatomical sites with the humerus and tail being more surgically challenging. The microstructural analysis using micro-computed tomography showed a significantly lower bone volume fraction, bone mineral density, and trabecular thickness of the humerus and iliac crest compared to the femur, tibia, and tail vertebrae. The harvested weight and volume did not differ between the donor sites. All donor sites apart from the femur yielded primary osteogenic cells confirmed by the presence of alkaline phosphatase and Alizarin Red S stain. Bone samples from the iliac crest showed the most consistent outgrowth of osteoprogenitor cells. In conclusion, the tibia and iliac crest may be the most favorable donor sites considering the surgical approach. However, due to the differences in microstructure of the cancellous bone and the consistency of outgrowth of osteoprogenitor cells, the donor sites may have different healing properties, that need further investigation in an in vivo study.

Standard in vitro evaluations of engineered bone substitutes are not sufficient to predict in vivo preclinical model outcomes.

Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non-unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, human bone marrow - derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with relevant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the second and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immunocompetent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced upregulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone formation. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies. STATEMENT OF SIGNIFICANCE: Preclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor correlation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.

Augmented screwdrivers can increase the performance of orthopaedic surgeons compared with use of normal screwdrivers.

Orthopaedic screws insertion can be trivialised as a simple procedure, however it is frequently performed poorly. Limited work exists defining how well surgeons insert screws or whether augmented screwdrivers can aid surgeons to reduce stripping rates and optimise tightness. We aimed to establish the performance of surgeons inserting screws and whether this be improved with screwdriver augmentation. 302 orthopaedic surgeons tightened 10 non-locking screws to what they determined to be optimum tightness into artificial bone sheets. The confidence in the screw purchase was given (1-10). A further 10 screws were tightened, using an augmented screwdriver that indicated when a predetermined optimum tightness was reached. The tightness for unstripped insertions under normal conditions and with the augmented screwdriver were 81% (95% CI 79-82%)(n = 1275) and 70% (95% CI 69-72%)(n = 2577) (p < 0.001). The stripping rates were 58% (95% CI 54-61%) and 15% (95% CI 12-17%) respectively (p < 0.001). The confidences when using the normal and augmented screwdrivers respectively were 7.2 and 7.1 in unstripped insertions and 6.2 and 6.5 in stripped insertions. Performance improved with an augmented screwdriver, both in reduced stripping rates and greater accuracy in detecting stripping. Augmenting screwdrivers to indicate optimum tightness offer potentially enormous clinical benefits by improving screw fixation.

Adaptive local thresholding can enhance the accuracy of HR-pQCT-based trabecular bone morphology assessment.

High-resolution peripheral quantitative computed tomography (HR-pQCT) devices can scan extremities at bone microstructural level in vivo and are used mainly in research of bone diseases. Two HR-pQCT scanners are commercially available to date: XtremeCT (first generation) and XtremeCT-II (second generation) from Scanco Medical AG (Switzerland). Recently, we have proposed an adaptive local thresholding (AT) technique and showed that it can improve quantification accuracy of bone microstructural parameters, with visually less sharp cone-beam CT (CBCT) images providing a similar accuracy than XtremeCT. The aim of this study was to evaluate whether the AT segmentation technique could enhance the accuracy of HR-pQCT in quantifying bone microstructural images and to assess whether the agreement between XtremeCT and XtremeCT-II could be improved. Nineteen radii were scanned with three scanners from Scanco Medical AG: (1) XtremeCT at 82 µm, (2) XtremeCT-II at 60.7 µm and (3) the small animal microCT scanner VivaCT40 at 19 µm voxel size. The scans were segmented applying two different methods, once following the manufacturer standard technique (ST), and once by means of AT. Three-dimensional (3D) morphological analysis was performed on the trabecular volume of the segmented images using the manufacturer's standard software to calculate bone volume fraction (BV/TV), trabecular thickness (Tb.Th), separation (Tb.Sp) and number (Tb.N). The average accuracy of XtremeCT improved from R2 = 0.76 (ST) to 0.85 (AT) and reached the same level of accuracy as XtremeCT-II with ST (R2 = 0.86). The largest improvements were obtained for BV/TV and Tb.Th. For XtremeCT-II, mean accuracy improved slightly from R2 = 0.86 (ST) to 0.89 (AT). For both segmentations and both scanners, the standard section was quantified slightly more accurate than the subchondral section. The agreement between the scanners was enhanced from R2 = 0.89 (ST) to 0.98 (AT). In conclusion, AT can enhance the accuracy of XtremeCT to quantify distal radius bone microstructural parameters close to XtremeCT-II level and increases the agreement between the two HR-pQCT scanners. High-resolution peripheral quantitative computed tomography, segmentation, bone microstructural parameters.

Advanced CT visualization improves the accuracy of orthopaedic trauma surgeons and residents in classifying proximal humeral fractures: a feasibility study.

PURPOSE: Osteosynthesis of proximal humeral fractures remains challenging with high reported failure rates. Understanding the fracture type is mandatory in surgical treatment to achieve an optimal anatomical reduction. Therefore, a better classification ability resulting in improved understanding of the fracture pattern is important for preoperative planning. The purpose was to investigate the feasibility and added value of advanced visualization of segmented 3D computed tomography (CT) images in fracture classification. METHODS: Seventeen patients treated with either plate-screw-osteosynthesis or shoulder hemi-prosthesis between 2015 and 2019 were included. All preoperative CT scans were segmented to indicate every fracture fragment in a different color. Classification ability was tested in 21 orthopaedic residents and 12 shoulder surgeons. Both groups were asked to classify fractures using three different modalities (standard CT scan, 3D reconstruction model, and 3D segmented model) into three different classification systems (Neer, AO/OTA and LEGO). RESULTS: All participants were able to classify the fractures more accurately into all three classification systems after evaluating the segmented three-dimensional (3D) models compared to both 2D slice-wise evaluation and 3D reconstruction model. This finding was significant (p < 0.005) with an average success rate of 94%. The participants experienced significantly more difficulties classifying fractures according to the LEGO system than the other two classifications. CONCLUSION: Segmentation of CT scans added value to the proximal humeral fracture classification, since orthopaedic surgeons were able to classify fractures significantly better into the AO/OTA, Neer, and LEGO classification systems compared to both standard 2D slice-wise evaluation and 3D reconstruction model.

Using embedded alginate microparticles to tune the properties of in situ forming poly(N-isopropylacrylamide)-graft-chondroitin sulfate bioadhesive hydrogels for replacement and repair of the nucleus pulposus of the intervertebral disc.

Low back pain is a major public health issue associated with degeneration of the intervertebral disc (IVD). The early stages of degeneration are characterized by the dehydration of the central, gelatinous portion of the IVD, the nucleus pulposus (NP). One possible treatment approach is to replace the NP in the early stages of IVD degeneration with a hydrogel that restores healthy biomechanics while supporting tissue regeneration. The present study evaluates a novel thermosensitive hydrogel based on poly(N-isopropylacrylamide-graft-chondroitin sulfate) (PNIPAAM-g-CS) for NP replacement. The hypothesis was tested that the addition of freeze-dried, calcium crosslinked alginate microparticles (MPs) to aqueous solutions of PNIPAAm-g-CS would enable tuning of the rheological properties of the injectable solution, as well as the bioadhesive and mechanical properties of the thermally precipitated composite gel. Further, we hypothesized that the composite would support encapsulated cell viability and differentiation. Structure-material property relationships were evaluated by varying MP concentration and diameter. The addition of high concentrations (50 mg/mL) of small MPs (20 ± 6 µm) resulted in the greatest improvement in injectability, compressive mechanical properties, and bioadhesive strength of PNIPAAm-g-CS. This combination of PNIPAAM-g-CS and alginate MPs supported the survival, proliferation, and differentiation of adipose derived mesenchymal stem cells toward an NP-like phenotype in the presence of soluble GDF-6. When implanted ex vivo into the intradiscal cavity of degenerated porcine IVDs, the formulation restored the compressive and neutral zone stiffnesses to intact values and resisted expulsion under lateral bending. Overall, results indicate the potential of the hydrogel composite to serve as a scaffold for supporting NP regeneration. This work uniquely demonstrates that encapsulation of re-hydrating polysaccharide-based MPs may be an effective method for improving key functional properties of in situ forming hydrogels for orthopedic tissue engineering applications.

Humanized Mice Exhibit Exacerbated Abscess Formation and Osteolysis During the Establishment of Implant-Associated Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the predominant pathogen causing osteomyelitis. Unfortunately, no immunotherapy exists to treat these very challenging and costly infections despite decades of research, and numerous vaccine failures in clinical trials. This lack of success can partially be attributed to an overreliance on murine models where the immune correlates of protection often diverge from that of humans. Moreover, S. aureus secretes numerous immunotoxins with unique tropism to human leukocytes, which compromises the targeting of immune cells in murine models. To study the response of human immune cells during chronic S. aureus bone infections, we engrafted non-obese diabetic (NOD)-scid IL2Rγnull (NSG) mice with human hematopoietic stem cells (huNSG) and analyzed protection in an established model of implant-associated osteomyelitis. The results showed that huNSG mice have increases in weight loss, osteolysis, bacterial dissemination to internal organs, and numbers of Staphylococcal abscess communities (SACs), during the establishment of implant-associated MRSA osteomyelitis compared to NSG controls (p < 0.05). Flow cytometry and immunohistochemistry demonstrated greater human T cell numbers in infected versus uninfected huNSG mice (p < 0.05), and that T-bet+ human T cells clustered around the SACs, suggesting S. aureus-mediated activation and proliferation of human T cells in the infected bone. Collectively, these proof-of-concept studies underscore the utility of huNSG mice for studying an aggressive form of S. aureus osteomyelitis, which is more akin to that seen in humans. We have also established an experimental system to investigate the contribution of specific human T cells in controlling S. aureus infection and dissemination.

High-Resolution Cone-Beam Computed Tomography is a Fast and Promising Technique to Quantify Bone Microstructure and Mechanics of the Distal Radius.

Obtaining high-resolution scans of bones and joints for clinical applications is challenging. HR-pQCT is considered the best technology to acquire high-resolution images of the peripheral skeleton in vivo, but a breakthrough for widespread clinical applications is still lacking. Recently, we showed on trapezia that CBCT is a promising alternative providing a larger FOV at a shorter scanning time. The goals of this study were to evaluate the accuracy of CBCT in quantifying trabecular bone microstructural and predicted mechanical parameters of the distal radius, the most often investigated skeletal site with HR-pQCT, and to compare it with HR-pQCT. Nineteen radii were scanned with four scanners: (1) HR-pQCT (XtremeCT, Scanco Medical AG, @ (voxel size) 82 µm), (2) HR-pQCT (XtremeCT-II, Scanco, @60.7 µm), (3) CBCT (NewTom 5G, Cefla, @75 µm) reconstructed and segmented using in-house developed software and (4) microCT (VivaCT40, Scanco, @19 µm-gold standard). The following parameters were evaluated: predicted stiffness, strength, bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), separation (Tb.Sp) and number (Tb.N). The overall accuracy of CBCT with in-house optimized algorithms in quantifying bone microstructural parameters was comparable (R2 = 0.79) to XtremeCT (R2 = 0.76) and slightly worse than XtremeCT-II (R2 = 0.86) which were both processed with the standard manufacturer's technique. CBCT had higher accuracy for BV/TV and Tb.Th but lower for Tb.Sp and Tb.N compared to XtremeCT. Regarding the mechanical parameters, all scanners had high accuracy (R2 [Formula: see text] 0.96). While HR-pQCT is optimized for research, the fast scanning time and good accuracy renders CBCT a promising technique for high-resolution clinical scanning.

Quantification of 3D microstructural parameters of trabecular bone is affected by the analysis software.

Over the last decades, the use of high-resolution imaging systems to assess bone microstructural parameters has grown immensely. Yet, no standard defining the quantification of these parameters exists. It has been reported that different voxel size and/or segmentation techniques lead to different results. However, the effect of the evaluation software has not been investigated so far. Therefore, the aim of this study was to compare the bone microstructural parameters obtained with two commonly used commercial software packages, namely IPL (Scanco, Switzerland) and CTan (Bruker, Belgium). We hypothesized that even when starting from the same segmented scans, different software packages will report different results. Nineteen trapezia and nineteen distal radii were scanned at two resolutions (20 µm voxel size with microCT and HR-pQCT 60 µm). The scans were segmented using the scanners' default protocol. The segmented images were analyzed twice, once with IPL and once with CTan, to quantify bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N) and specific bone surface (BS/BV). Only small differences between IPL and CTan were found for BV/TV. For Tb.Th, Tb.Sp and BS/BV high correlations (R2 ≥ 0.99) were observed between the two software packages, but important relative offsets were observed. For microCT scans, the offsets were relative constant, e.g., around 15% for Tb.Th. However, for the HR-pQCT scans the mean relative offsets ranged over the different bone samples (e.g., for Tb.Th from 14.5% to 19.8%). For Tb.N, poor correlations (0.43 ≤ R2 ≤ 0.81) for all tested cases were observed. We conclude that trabecular bone microstructural parameters obtained with IPL and CTan cannot be directly compared except for BV/TV. For Tb.Th, Tb.Sp and BS/BV, correction factors can be determined, but these depend on both the image voxel size and specific anatomic location. The two software packages did not produce consistent data on Tb.N. The development of a universal standard seems desirable.

Correlation Between Cone-Beam Computed Tomography and High-Resolution Peripheral Computed Tomography for Assessment of Wrist Bone Microstructure.

High-resolution peripheral quantitative computed tomography (HR-pQCT) is considered as the best technique to measure bone microarchitecture in vivo. However, a breakthrough for medical applications is inhibited because of the restricted field of view (∼9 mm) and a relatively long acquisition time (∼3 minutes). The goal of this study was to compare the accuracy of cone-beam computed tomography (CBCT) and HR-pQCT and to determine the agreement between CBCT and HR-pQCT in quantifying bone structural parameters. Nineteen trapezia of arthritic patients were scanned four times ex vivo: 1) CBCT (NewTom 5G, Cefla, at 75 µm); 2) HR-pQCT (XTremeCT-I, Scanco, at 82 µm); 3) HR-pQCT (XTremeCT-II, Scanco, at 60.7 µm); and 4) microCT (SkyScan1172, Bruker, at 19.84 µm). XTremeCT-I and XtremeCT-II were reconstructed, segmented, and analyzed following the manufacturer's guidelines. CBCT was reconstructed with in-house developed software and analyzed twice: once with an adaptive segmentation technique combined with a direct analysis method (AT-DM) and once with a Laplace-Hamming filtering technique combined with an indirect analysis method (LH-IM). Parameters of interest included bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), separation (Tb.Sp), and number (Tb.N). The analyses of the CBCT data showed that the AT-DM analysis correlated better with microCT for BV/TV, Tb.Sp, and Tb.N, whereas the LH-IM technique correlated better for Tb.Th. Evaluated over all parameters, the coefficient of determination for XtremeCT-I, XtremeCT-II, and CBCT were higher as R2 = 0.68, 0.72, and 0.67, respectively. For CBCT, the correlations improved when three samples with very thin trabeculae close to each other were excluded and became similar to those for XtremeCT-I and XtremeCT-II. Interesting for clinical practice is that those bones could be identified automatically with the CBCT scanner. We conclude that CBCT produced similar accuracy as HR-pQCT in bone morphometric analyses of trapezia. The broader range of application, larger field of view, and shorter acquisition time make CBCT a valuable alternative to HR-pQCT. © 2019 American Society for Bone and Mineral Research.

Quantification of bone microstructure in the wrist using cone-beam computed tomography.

Due to the rising life expectancy, bone diseases (e.g. osteoporosis, osteoarthritis) and trauma (e.g. fracture) have become an important socio-economic burden. Accurate visualization and quantification of the bone microstructure in vivo is seen as an important step to enhance diagnosis and treatment. Micro-computed tomography (microCT) has become the gold standard in three-dimensional (3D) imaging of trabecular bone structure. Yet, usage is limited to ex vivo analyses, hence, it cannot be used to evaluate bone and bone adaptive responses in a patient. High-resolution peripheral computed tomography (HR-pQCT) is considered the best technique to measure the bone microarchitecture in vivo. By design HR-pQCT is limited to scanning extremities, such as the distal radius and distal tibia with a limited field of view and long scanning time (~2 à 3 min. for a stack of 0.9 cm). Cone-beam computed tomography (CBCT) is a promising alternative with a much larger field of view. Yet, CBCT is challenged by artefacts that reduce image contrast, such that it is currently being used for qualitative evaluation only. Therefore, the aims of this work were first to enhance image contrast and second to determine the accuracy of high-resolution CBCT for bone microarchitectural assessment. Trapezia of nineteen female arthritic patients were scanned twice ex vivo; once using CBCT (NewTom 5G, Cefla, Verona, Italy) at a nominal voxel size of 75 µm and once using microCT (SkyScan 1172, Bruker, Kontich, Belgium) at a voxel size of 19.84 µm. The CBCT-scans were reconstructed following 2 protocols: (1) using the commercial software delivered with the scanner and (2) using in-house developed software. After reconstruction and image processing, the images were segmented using adaptive thresholding. Bone morphometric parameters including bone volume (BV), total tissue volume (TV), bone volume fraction (BV/TV), bone surface density (BS/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) were calculated. Statistical evaluations were made at a significance level of 5%. Significant correlations were found between the CBCT-based bone parameters and the microCT-based parameters with R2 > 0.68 The in-house reconstructed software outperformed the commercial software. Smaller bias (overestimation of Tb.Th decreased from 114.24% to 59.96%) as well as higher correlations were observed for the in-house processed images. Still, a significant overestimation was observed for BV/TV and Tb. Th and an underestimation for Tb.N. We conclude that our CBCT image reconstruction improved image contrast which allowed for an accurate quantification of trabecular bone microarchitecture.