Leptin receptor gene deficiency minimally affects osseointegration in rats.

Metabolic syndrome represents a cluster of conditions such as obesity, hyperglycaemia, dyslipidaemia, and hypertension that can lead to type 2 diabetes mellitus and/or cardiovascular disease. Here, we investigated the influence of obesity and hyperglycaemia on osseointegration using a novel, leptin receptor-deficient animal model, the Lund MetS rat. Machined titanium implants were installed in the tibias of animals with normal leptin receptor (LepR+/+) and those harbouring congenic leptin receptor deficiency (LepR-/-) and were left to heal for 28 days. Extensive evaluation of osseointegration was performed using removal torque measurements, X-ray micro-computed tomography, quantitative backscattered electron imaging, Raman spectroscopy, gene expression analysis, qualitative histology, and histomorphometry. Here, we found comparable osseointegration potential at 28 days following implant placement in LepR-/- and LepR+/+ rats. However, the low bone volume within the implant threads, higher bone-to-implant contact, and comparable biomechanical stability of the implants point towards changed bone formation and/or remodelling in LepR-/- rats. These findings are corroborated by differences in the carbonate-to-phosphate ratio of native bone measured using Raman spectroscopy. Observations of hypermineralised cartilage islands and increased mineralisation heterogeneity in native bone confirm the delayed skeletal development of LepR-/- rats. Gene expression analyses reveal comparable patterns between LepR-/- and LepR+/+ animals, suggesting that peri-implant bone has reached equilibrium in healing and/or remodelling between the animal groups.

Bone structure and composition in a hyperglycemic, obese, and leptin receptor-deficient rat: Microscale characterization of femur and calvarium.

Metabolic abnormalities, such as diabetes mellitus and obesity, can impact bone quantity and/or bone quality. In this work, we characterize bone material properties, in terms of structure and composition, in a novel rat model with congenic leptin receptor (LepR) deficiency, severe obesity, and hyperglycemia (type 2 diabetes-like condition). Femurs and calvaria (parietal region) from 20-week-old male rats are examined to probe bones formed both by endochondral and intramembranous ossification. Compared to the healthy controls, the LepR-deficient animals display significant alterations in femur microarchitecture and in calvarium morphology when analyzed by micro-computed X-ray tomography (micro-CT). In particular, shorter femurs with reduced bone volume, combined with thinner parietal bones and shorter sagittal suture, point towards a delay in the skeletal development of the LepR-deficient rodents. On the other hand, LepR-deficient animals and healthy controls display analogous bone matrix composition, which is assessed in terms of tissue mineral density by micro-CT, degree of mineralization by quantitative backscattered electron imaging, and various metrics extrapolated from Raman hyperspectral images. Some specific microstructural features, i.e., mineralized cartilage islands in the femurs and hyper-mineralized areas in the parietal bones, also show comparable distribution and characteristics in both groups. Overall, the altered bone microarchitecture in the LepR-deficient animals indicates compromised bone quality, despite the normal bone matrix composition. The delayed development is also consistent with observations in humans with congenic Lep/LepR deficiency, making this animal model a suitable candidate for translational research.

Development of Highly Sensitive Digital Droplet PCR for Detection of cKIT Mutations in Circulating Free DNA That Mediate Resistance to TKI Treatment for Gastrointestinal Stromal Tumor (GIST).

BACKGROUND: Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). METHODS: We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. RESULTS: Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. CONCLUSIONS: This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.

Bone without borders - Monetite-based calcium phosphate guides bone formation beyond the skeletal envelope.

Calcium phosphates (CaP) represent an important class of osteoconductive and osteoinductive biomaterials. As proof-of-concept, we show how a multi-component CaP formulation (monetite, beta-tricalcium phosphate, and calcium pyrophosphate) guides osteogenesis beyond the physiological envelope. In a sheep model, hollow dome-shaped constructs were placed directly over the occipital bone. At 12 months, large amounts of bone (∼75%) occupy the hollow space with strong evidence of ongoing remodelling. Features of both compact bone (osteonal/osteon-like arrangements) and spongy bone (trabeculae separated by marrow cavities) reveal insights into function/need-driven microstructural adaptation. Pores within the CaP also contain both woven bone and vascularised lamellar bone. Osteoclasts actively contribute to CaP degradation/removal. Of the constituent phases, only calcium pyrophosphate persists within osseous (cutting cones) and non-osseous (macrophages) sites. From a translational perspective, this multi-component CaP opens up exciting new avenues for osteotomy-free and minimally-invasive repair of large bone defects and augmentation of the dental alveolar ridge.

Complex geometry and integrated macro-porosity: Clinical applications of electron beam melting to fabricate bespoke bone-anchored implants.

The last decade has witnessed rapid advancements in manufacturing technologies for biomedical implants. Additive manufacturing (or 3D printing) has broken down major barriers in the way of producing complex 3D geometries. Electron beam melting (EBM) is one such 3D printing process applicable to metals and alloys. EBM offers build rates up to two orders of magnitude greater than comparable laser-based technologies and a high vacuum environment to prevent accumulation of trace elements. These features make EBM particularly advantageous for materials susceptible to spontaneous oxidation and nitrogen pick-up when exposed to air (e.g., titanium and titanium-based alloys). For skeletal reconstruction(s), anatomical mimickry and integrated macro-porous architecture to facilitate bone ingrowth are undoubtedly the key features of EBM manufactured implants. Using finite element modelling of physiological loading conditions, the design of a prosthesis may be further personalised. This review looks at the many unique clinical applications of EBM in skeletal repair and the ground-breaking innovations in prosthetic rehabilitation. From a simple acetabular cup to the fifth toe, from the hand-wrist complex to the shoulder, and from vertebral replacement to cranio-maxillofacial reconstruction, EBM has experienced it all. While sternocostal reconstructions might be rare, the repair of long bones using EBM manufactured implants is becoming exceedingly frequent. Despite the various merits, several challenges remain yet untackled. Nevertheless, with the capability to produce osseointegrating implants of any conceivable shape/size, and permissive of bone ingrowth and functional loading, EBM can pave the way for numerous fascinating and novel applications in skeletal repair, regeneration, and rehabilitation. STATEMENT OF SIGNIFICANCE: Electron beam melting (EBM) offers unparalleled possibilities in producing contaminant-free, complex and intricate geometries from alloys of biomedical interest, including Ti6Al4V and CoCr. We review the diverse range of clinical applications of EBM in skeletal repair, both as mass produced off-the-shelf implants and personalised, patient-specific prostheses. From replacing large volumes of disease-affected bone to complex, multi-material reconstructions, almost every part of the human skeleton has been replaced with an EBM manufactured analog to achieve macroscopic anatomical-mimickry. However, various questions regarding long-term performance of patient-specific implants remain unaddressed. Directions for further development include designing personalised implants and prostheses based on simulated loading conditions and accounting for trabecular bone microstructure with respect to physiological factors such as patient's age and disease status.

The impact of medication on osseointegration and implant anchorage in bone determined using removal torque-A review.

Permanently anchored metal implants are frequently used in dental, craniomaxillofacial, and orthopaedic rehabilitation. The success of such therapies is owed to the phenomenon of osseointegration-the direct connection between the living bone and the implant. The extent of biomechanical anchorage (i.e., physical interlocking between the implant and bone) can be assessed with removal torque (RTQ) measurement. Implant anchorage is strongly influenced by underlying bone quality, involving physicochemical and biological properties such as composition and structural organisation of extracellular matrix, extent of micro-damage, and bone turnover. In this review, we evaluated the impact of various pharmacological agents on osseointegration, from animal experiments conducting RTQ measurements. In addition to substances whose antiresorptive and/or anti-catabolic effects on bone are well-documented (e.g., alendronate, zoledronate, ibandronate, raloxifene, human parathyroid hormone, odanacatib, and the sclerostin monoclonal antibody), positive effects on RTQ have been reported for substances that do not primarily target bone (e.g., aminoguanidine, insulin, losartan, simvastatin, bone morphogenetic protein, alpha-tocopherol, and the combination of silk fibroin powder and platelet-rich fibrin). On the contrary, several substances (e.g., prednisolone, cyclosporin A, cisplatin, and enamel matrix derivative) tend to adversely impact RTQ. While morphometric parameters such as bone-implant contact appear to influence the biomechanical anchorage, increased or decreased RTQ is not always accompanied by corresponding fluctuations in bone-implant contact. This further confirms that factors such as bone quality underpin biomechanical anchorage of metal implants. Several fundamental questions on drug metabolism and bioavailability, drug dosage, animal-to-human translation, and the consequences of treatment interruption remain yet unanswered.

Early assessment of circulating tumor DNA after curative-intent resection predicts tumor recurrence in early-stage and locally advanced non-small-cell lung cancer.

Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non-small-cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted-capture high-throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early-stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative-intent tumor resection (median follow-up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1-2 weeks postsurgery, and during follow-up. Deep next-generation sequencing using unique molecular identifiers was performed to identify and quantify tumor-specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL-1 , respectively). Four patients (19%) remained ctDNA-positive at 1-2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA-negative patients (33%) after surgery experienced relapse during follow-up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression-free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early-stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse.

Personalized Treatment Selection and Disease Monitoring Using Circulating Tumor DNA Profiling in Real-World Cancer Patient Management.

BACKGROUND: Circulating tumor DNA (ctDNA) in the blood plasma of cancer patients is an emerging biomarker used across oncology, facilitating noninvasive disease monitoring and genetic profiling at various disease milestones. Digital droplet PCR (ddPCR) technologies have demonstrated high sensitivity and specificity for robust ctDNA detection at relatively low costs. Yet, their value for ctDNA-based management of a broad population of cancer patients beyond clinical trials remains elusive. METHODS: We developed mutation-specific ddPCR assays that were optimized for their use in real-world cancer management, covering 12 genetic aberrations in common cancer genes, such as EGFR, BRAF, KIT, KRAS, and NRAS. We assessed the limit of detection (LOD) and the limit of blank (LOB) for each assay and validated their performance for ctDNA detection using matched tumor sequencing. RESULTS: We applied our custom ddPCR assays to 352 plasma samples from 96 patients with solid tumors. Mutation detection in plasma was highly concordant with tumor sequencing, demonstrating high sensitivity and specificity across all assays. In 20 cases, radiographic cancer progression was mirrored by an increase of ctDNA concentrations or the occurrence of novel mutations in plasma. Moreover, ctDNA profiling at diagnosis and during disease progression reflected personalized treatment selection through the identification of actionable gene targets in 20 cases. CONCLUSION: Collectively, our work highlights the potential of ctDNA assessment by sensitive ddPCR for accurate disease monitoring, robust identification of resistance mutations, and upfront treatment selection in patients with solid tumors. We envision an increasing future role for ctDNA profiling within personalized cancer management in daily clinical routine.