Host Bone Research Articles

The Effects of Structural & Materials Design on the Mechanisms of Tissue Integration with the 3D Printed Polyether-Ether-Ketone Cranial Implants in Vivo

3D printed polyether-ether-ketone (PEEK) implant has become a popular clinical alternative to implants made of titanium alloy for cranial bone substitutes due to its bone-match properties, however its biological inert hindered the progress of clinical applications of such material. To enhance the tissue integration capability of PEEK implants and promote their long-term stability, cranial implants of gradient porous structures were designed and manufactured via fused filament fabrication (FFF) 3D printing technology in both PEEK and PEEK/HA composites materials, then functionally evaluation of the implants on the tissue in-growth and the osteointegration mechanisms was conducted via animal tests. The 3D printed PEEK scaffold was found to have 2–5 folds of the compressive strength to those of the natural cranial bone. The in vivo studies verified that the porous PEEK/HA scaffold could effectively induce the bone ingrowth to form a stable biointegration boundary surrounding the host bone tissue after 4 weeks of implantation. Moreover, the PEEK/HA scaffold showed no significant advantages in improving the soft tissue in-growth, only making its distribution more evenly. It is also interestingly to find out that the vertically connective pores throughout the implant did not enhance the tissue binding force even though it did promote the nutrient transportation. In conclusion, the use of PEEK/HA composite material and a well-designed porous structure was proved to be an effective approach to improve the biointegration between the implant and host tissues.

Mechanical load applied by Intraosseous Transcutaneous Amputation Prosthesis (ITAP) during walking on level and sloped treadmill: A case study

This proof of concept study presents a method to collect and analyse kinetic data from one participant with a transfemoral amputation fitted with a percutaneous osseointegrated implant walking on a level and sloped treadmill. We describe the construction of and results from a bespoke wireless six axis load cell built into one participant's prosthetic assembly. The load cell does not clinically compromise the participant in any way and is an initial milestone in the development of a light-weight wireless load cell for use with percutaneous osseointegrated implants. In this case, it is the first time that kinetic data from a participant fitted with an Intraosseous Transcutaneous Amputation Prosthesis has been published. We propose that the data can be used to model the load transfer to the host bone, with several clinically significant applications. The raw dynamic data are made available and quasi-static load cases for each functional phase of gait are presented. Peak forces obtained in the medio-lateral (X), cranio-caudal (Y) and antero-posterior (Z) axes over level ground respectively were -243.8 N (0.24 BW), 1321.5 N (1.31 BW) and -421.8 N (0.42 BW); uphill were -141.0 N (0.14 BW), 1604.2 N (1.59 BW), -498.1 N (0.49 BW); downhill were -206.0 N (0.20 BW), 1103.9 N (1.09 BW), -547.2 N (0.54 BW). The kinetics broadly followed able bodied gait patterns with some gait strategies consistent in participants with other implant designs or prosthetic socket connections, for example offloading the artificial limb downhill.

In vitro evaluation of BMSCs early proliferation on minocycline-loaded electrospun nanofibers membrane.

Electrospun nanofibers could simulate the natural extracellular matrix (ECM) of the host bone, while minocycline (MINO) is a broad-spectrum tetracycline antibiotic which has been found to have multiple non-antibiotics biological effects that promotes osteogenesis in vitro and in vivo. The present study aims at constructing a polylactic acid (PLA) electrospun nanofiber membrane loaded with MINO to enhance Bone marrow mesenchymal stem cells (BMSCs) adhesion and proliferation for early clinical treatment. The MINO-PLA membrane were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR) and in vitro drug release study. The antibacterial ability was also investigated. In addition, in vitro cellular proliferation experiment was performed to verify whether the PLA electrospun nanofibers membrane loaded with MINO enhance BMSCs adhesion and proliferation. Analyzing the drug release and cell growth results, it was found that only the effective concentration of MINO-PLA could help the growth of BMSCs in the short term. This is related to the drug release rate of MINO-PLA and the initial concentration of MINO. This study shows that by controlling the concentration and release rate of MINO with electrospinning PLA, BMSCs could proliferate on it, and a new bone repair material had been made in this study.

UNMET CLINICAL NEEDS AND CHALLENGES IN ORTHOPAEDIC IMPLANTS

Infections are among the most diffused complications of the implantation of medical devices. In orthopedics, they pose severe societal and economic burden and interfere with the capability of the implants to integrate in the host bone, significantly increasing failure risk. Infection is particularly severe in the case of comorbidities and especially bone tumors, since oncologic patients are fragile, have higher infection rate and impaired osteoregenerative capabilities. For this reason, prevention of infection is to be preferred over treatment.This is even more important in the case of spine surgery, since spine is among the main site for tumor metastases and because incidence of post operative surgical-site infections is significant (up to 15-20%) and surgical options are limited by the need of avoiding damaging the spinal cord.Functionalization of the implant surfaces, so as to address infection and, possibly, co- adjuvate anti-tumor treatments, appears as a breakthrough innovation. Unmet clinical needs in infection and tumors is presented, with a specific focus on the spine, then, new perspectives are highlighted for their treatment.

INFLUENCE OF FLUID UPTAKE ON THE MECHANICAL AND THERMAL PROPERTIES OF PMMA-BASED BONE CEMENT

According to the latest report from the German Arthroplasty Registry, aseptic loosening is the primary cause of implant failure following primary hip arthroplasty. Osteolysis of the proximal femur due to the stress-shielding of the bone by the implant causes loss of fixation of the proximal femoral stem, while the distal stem remains fixed.Removing a fixed stem is a challenging process. Current removal methods rely on manual tools such as chisels, burrs, osteotomes, drills and mills, which pose the risk of bone fracture and cortical perforation. Others such as ultrasound and laser, generate temperatures that could cause thermal injury to the surrounding tissues and bone. It is crucial to develop techniques that preserve the host bone, as its quality after implant removal affects the outcome of a revision surgery.A gentler removal method based on the transcutaneous heating of the implant by induction is proposed. By reaching the glass transition temperature (TG) of the periprosthetic cement, the cement is expected to soften, enabling the implant to be gently pulled out. The in-vivo environment comprises body fluids and elevated temperatures, which deteriorate the inherent mechanical properties of bone cement, including its TG. We aimed to investigate the effect of fluid absorption on the TG (ASTM E2716-09) and Vicat softening temperature (VST) (ISO 306) of Palacos R cement (Heraeus Medical GmbH) when dry and after storage in Ringer's solution for up to 8 weeks.Samples stored in Ringer's solution exhibited lower TG and VST than those stored in air. After 8 weeks, the TG decreased from 95.2°C to 81.5°C in the Ringer's group, while the VST decreased from 104.4°C to 91.9°C. These findings will be useful in the ultimate goal of this project which is to design an induction-based system for implant removal.Acknowledgements: Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB/TRR-298-SIIRI – Project-ID 426335750

3D-PRINTED ABSORBABLE METALLIC MEDICAL DEVICES AND THEIR APPLICATION POTENTIAL

AM specifically allows for cost-efficient production of patient-specific Orthopaedic medical devices with unusual designs and properties. A porous design allows to adjust the stiffness of metallic implants to that of the host bone. Beyond traditional metals, like titanium alloys, this talk will review the present state-of-the-art of directly printed absorbable metal families. Physicochemical, mechanical and biological properties of standardized design prototypes from all currently available metal families will be compared and their clinical application potential discussed. The impact of in vitro test environments on comparative corrosion behavior, post manufacturing aspects, and the recent status quo in biocompatibility testing and present knowledge gaps will be addressed.

Bone Regeneration Using an Alloplastic Graft Material that Combines β Tricalcium Phosphate and Calcium Sulphate: A Case Series Report with Histology

This retrospective case series reports on the use of an established alloplastic bone product composed of a 65:35 ratio of β Tricalcium Phosphate and Calcium Sulphate to facilitate host bone regeneration in a variety of intra-oral sites and clinical indications. The study involved seven patients treated by seven different clinicians operating at five geographical sites. All cases included trephined bone cores harvested at 12 and 33 weeks. These cores were subject to histological analysis using standardized protocols at six separate laboratories. The primary measured outcome was the percentage of residual graft, new bone, and connective tissue at the measured time points. The results of this case series suggest that the alloplastic bone product is 50% resorbed and replaced by new host bone at 12 weeks, with further consistent resorption to 85% at 33 weeks. These results suggest that this alloplastic product can be successfully used in a wide variety of intra-oral bone grafting protocols leading to consistent resorption of the material and its replacement with new host bone, independent of clinician and established histological analytical protocol used.

Clinical Use of a Mineral-Organic Resorbable Bone Adhesive: A First Human Case Report with a 3-Year Follow-up.

The ability for clinicians to adequately obtain primary stability in host bone is critical to the success of dental implants. Numerous conditions require dentists to perform multistage approaches to rebuild deficient bone volume prior to surgically placing implants. In many instances, implant placement cannot be achieved due to a lack of primary implant stability. Recently, a novel mineral-organic resorbable bone adhesive (MORBA) has demonstrated promising results in animal studies. MORBA is a synthetic, injectable, self-setting, load-bearing adhesive biomaterial that exhibits osteopromotive properties and bonds bone to bone and metal within 10 minutes and can fully resorb in 30 weeks. Its unique novel formulation was developed from biomimetic proteins found in marine animal creatures that possess distinct adhesive properties underwater. Excellent long-term results have shown its potential use for achieving primary stability in immediate implants. The present case report demonstrates the first use of MORBA in a human patient, utilized on a nonrestorable mandibular first molar. MORBA was utilized after placement of a mobile 5.8-mm implant to achieve stabilization. At 3 months postsurgery, both clinical and CBCT evaluations showed maintained implant stability. One year after implant placement, radiographic bone was seen on the buccal surface of the implant with continued long-term stabilization. This case report extends to 3 years whereby the use of MORBA, in an initially unstable situation, demonstrated an excellent long-term follow-up. MORBA provided immediate implant stability with resorbable characteristics, leading to successful long-term clinical outcomes up to 3 years. This innovative biomaterial offers a more efficient solution to a critical problem in implant dentistry, allowing optimal primary stability during immediate implant placement, thus reducing treatment times and costs.

Numerical evaluation of spinal reconstruction using a 3D printed vertebral body replacement implant: effects of material anisotropy.

Artificial vertebral implants have been widely used for functional reconstruction of vertebral defects caused by tumors or trauma. However, the evaluation of their biomechanical properties often neglects the influence of material anisotropy derived from the host bone and implant's microstructures. Hence, this study aims to investigate the effect of material anisotropy on the safety and stability of vertebral reconstruction. Two finite element models were developed to reflect the difference of material properties between linear elastic isotropy and nonlinear anisotropy. Their biomechanical evaluation was carried out under different load conditions including flexion, extension, lateral bending and axial rotation. These performances of two models with respect to safety and stability were analyzed and compared quantitatively based on the predicted von Mises stress, displacement and effective strain. The maximum von Mises stress of each component in both models was lower than the yield strength of respective material, while the predicted results of nonlinear anisotropic model were generally below to those of the linear elastic isotropic model. Furthermore, the maximum von Mises stress of natural vertebra and reconstructed system was decreased by 2-37MPa and 20-61MPa, respectively. The maximum reductions for the translation displacement of the artificial vertebral body implant and motion range of whole model were reached to 0.26mm and 0.77°. The percentage of effective strain elements on the superior and inferior endplates adjacent to implant was diminished by up to 19.7% and 23.1%, respectively. After comprehensive comparison, these results indicated that the finite element model with the assumption of linear elastic isotropy may underestimate the safety of the reconstruction system, while misdiagnose higher stability by overestimating the range of motion and bone growth capability.

Three-dimensional printed bioresorbable scaffold for maxillofacial bone reconstruction: A Scoping Review.

This scoping review aimed to provide an overview of current advancements in virtual planning and custom-made 3D-printed bioresorbable scaffolds, and to evaluate their clinical outcomes in maxillofacial reconstructive surgeries. Electronic searches of PubMed, EMBASE, Web of Science, Scopus, and Cochrane Library databases were conducted for publications up to June 2024. Included in the review were reports evaluating patients who underwent maxillofacial bone defect reconstruction using virtual planning and custom-made 3D-printed bioresorbable scaffolds. Data on postoperative complications, new bone formation, scaffold resorption, dental implant success/survival, and patient satisfaction were collected. The electronic search found 5799 results (3438 unique citations). A total of 54 studies were evaluated for full-text reading, of which 41 were excluded based on the inclusion criteria. Thirteen studies (6 case reports, 5 case series, one prospective clinical study and one randomized clinical trial) were included. These studies assessed the effectiveness of 3D-printed scaffolds in reconstructing maxillofacial defects, bone augmentation for dental implant placement, and regeneration of periosseous defects. Most of the 3D-printed scaffolds were biocompatible and did not cause local or systemic adverse events. However, some postoperative complications were reported, including graft exposure, wound dehiscence, and local infection. Overall, the 3D-printed scaffolds demonstrated favorable dimensional compatibility with deformities, provided durable support, promoted bone formation, achieved adequate bone union with host bone tissues, and supported dental implant placement without additional guided bone regeneration. In conclusion, custom-made 3D-printed bioresorbable scaffolds, guided by virtual planning, present a promising option for maxillofacial reconstruction due to their accuracy, osteoconductivity, and biocompatible properties.

Does the interface between individual 3D acetabular implants and host bone influence the functional outcomes in patients with severe bone loss after revision surgery?

There is a wide range of commercially produced revision implants for adequate reconstruction of acetabular large bone defects today, however, it is not always possible to achieve long-term survival of these implants. There is an increasing number of scientific publications concerning the use of custom-designed 3D components, which make it possible not only to achieve stable fixation and connect the pelvic bones but also to restore hip joint biomechanics. To evaluate the positioning of 3D acetabular implants after revision hip arthroplasty and its impact on clinical and functional outcomes. we analyzed results in 48 patients with bone defect types IIIA and IIIB Paprosky types, after revision hip arthroplasty. A prospective study was conducted from 2017 to 2023. Revision arthroplasty due to aseptic loosening of the components was performed in 30 cases and as a second stage of periprosthetic infection treatment in 18 cases. We did not achieve a statistically significant difference when using additional flanges and clinical and functional results. In 2 cases we faced aseptic loosening in patients using flanges. In no case were we able to install an implant with 100% adherence to porous structure compared to preoperatively planned adherence. According to the WOMAC and VAS scales, increasing the contact area of the components showed a slight statistical difference in the improvement of clinical and functional results and the reduction of pain. When acetabular 3D components adhered to the bone by more than 68%, we did not register a single complication in the postoperative period, and acetabular 3D components adhered to the bone by less than 68%, a total of 8 (16.6%) complications were registered.

New Horizons of Cementless Total Knee Arthroplasty.

Considering the increasing number of young and active patients needing TKA, orthopedic surgeons are looking for a long-lasting and physiological bond for the prosthetic implant. Multiple advantages have been associated with cementless fixation including higher preservation of the native bone stock, avoidance of cement debris with subsequent potential third-body wear, and the achievement of a natural bond and osseointegration between the implant and the bone that will provide a durable and stable fixation. Innovations in technology and design have helped modern cementless TKA implants to improve dramatically. Better coefficient of friction and reduced Young's modulus mismatch between the implant and host bone have been related to the use of porous metal surfaces. Moreover, biologically active coatings have been used on modern implants such as periapatite and hydroxyapatite. These factors have increased the potential for ingrowth by reducing micromotion and increasing osteoconductive properties. New materials with better biocompatibility, porosity, and roughness have been introduced to increase implant stability. Innovations in technology and design have helped modern cementless TKA implants improve primary stability in both the femur and tibia. This means that short-term follow-up are comparable to cemented. These positive prognostic factors may lead to a future in which cementless fixation may be considered the gold-standard technique in young and active patients.

Bone-inspired hydroxyapatite nanowire-based bioceramics with superior mechanical performance

The fabrication of hydroxyapatite (HAP)-based bioceramics with similar mechanical performance to bone by traditional sintering process is still challenge. In bone tissues, HAP mineralized collagen fibrils and fiber-oriented microstructure play key roles in superior mechanical performance of bone. Inspired by the fiber-oriented microstructure and biomineralization process of bone, the mineralized HAP-based bioceramics (M-HAP) are successfully fabricated via directional assembly and biomimetic mineralization strategies based on ultralong HAP nanowires. The M-HAP bioceramics show comparable mechanical performance to human cortical bone in terms of flexural strength, tensile strength, Young's modulus and hardness, which could overcome the issue of mismatched mechanical properties between traditional HAP implants and host bone. In addition, the M-HAP bioceramics also exhibit good cytocompatibility in vitro and no obvious inflammatory response after implantation in vivo. Such bone-inspired bioceramics with both high mechanical property and good biocompatibility have great potential in the field of bone regeneration.

Engineered Cell‐Laden Armor Unit‐Mimicking Bioceramic Granules for Bone Regeneration

AbstractBioceramic granules have been used as fillers for the repair of various bone defects, but structural instability leads to susceptibility to displacement or dislodgement in practice. Armor unit is a concrete block placed along the breakwater to resist waves and protect the dike. Inspired by this, an armor unit‐mimicking (AU) bioceramic granule by three‐dimentional (3D) printing is developed, and combine it with adipose‐derived stem cells (ADSCs) overexpressing fibroblast growth factor 23 (FGF23) to construct tissue‐engineered bone. In vitro results show that AU granules and FGF23 transfection synergistically promote osteogenic differentiation of ADSCs. For the repair of beagle femoral metaphyseal defects, the implanted AU granules are stacked and interlocked to form an impact‐resistant porous scaffold. With the assistance of ADSCsFGF23, AU granules exhibit enhanced new bone formation and host bone maintenance. Taken together, these findings demonstrate the potential of 3D‐printed AU granules in combination with FGF23‐overexpressing ADSCs as a novel bone void filler for ease of handling and enhanced bone regeneration.

Design of 3D-printed prostheses for reconstruction of periacetabular bone tumors using topology optimization.

Background: Prostheses for the reconstruction of periacetabular bone tumors are prone to instigate stress shielding. The purpose of this study is to design 3D-printed prostheses with topology optimization (TO) for the reconstruction of periacetabular bone tumors and to add porous structures to reduce stress shielding and facilitate integration between prostheses and host bone. Methods: Utilizing patient CT data, we constructed a finite element analysis (FEA) model. Subsequent phases encompassed carrying out TO on the designated area, utilizing the solid isotropic material penalization model (SIMP), and this optimized removal area was replaced with a porous structure. Further analyses included preoperative FEA simulations to comparatively evaluate parameters, including maximum stress, stress distribution, strain energy density (SED), and the relative micromotion of prostheses before and after TO. Furthermore, FEA based on patients' postoperative CT data was conducted again to assess the potential risk of stress shielding subsequent to implantation. Ultimately, preliminary follow-up findings from two patients were documented. Results: In both prostheses, the SED before and after TO increased by 143.61% (from 0.10322 to 0.25145mJ/mm3) and 35.050% (from 0.30964 to 0.41817mJ/mm3) respectively, showing significant differences (p < 0.001). The peak stress in the Type II prosthesis decreased by 10.494% (from 77.227 to 69.123MPa), while there was no significant change in peak stress for the Type I prosthesis. There were no significant changes in stress distribution or the proportion of regions with micromotion less than 28μm before and after TO for either prosthesis. Postoperative FEA verified results showed that the stress in the pelvis and prostheses remained at relatively low levels. The results of follow-up showed that the patients had successful osseointegration and their MSTS scores at the 12thmonth after surgery were both 100%. Conclusion: These two types of 3D-printed porous prostheses using TO for periacetabular bone tumor reconstruction offer advantages over traditional prostheses by reducing stress shielding and promoting osseointegration, while maintaining the original stiffness of the prosthesis. Furthermore, in vivo experiments show that these prostheses meet the requirements for daily activities of patients. This study provides a valuable reference for the design of future periacetabular bone tumor reconstruction prostheses.

Evaluation of Hard and Soft Tissue Responses to Four Different Generation Bioresorbable Materials-Poly-l-Lactic Acid (PLLA), Poly-l-Lactic Acid/Polyglycolic Acid (PLLA/PGA), Uncalcined/Unsintered Hydroxyapatite/Poly-l-Lactic Acid (u-HA/PLLA) and Uncalcined/Unsintered Hydroxyapatite/Poly-l-Lactic Acid/Polyglycolic Acid (u-HA/PLLA/PGA) in Maxillofacial Surgery: An In-Vivo Animal Study

Bone stabilization using osteosynthesis devices is essential in maxillofacial surgery. Owing to numerous disadvantages, bioresorbable materials are preferred over titanium for osteofixation in certain procedures. The biomaterials used for osteosynthesis in maxillofacial surgery have been subdivided into four generations. No study has compared the tissue responses generated by four generations of biomaterials and the feasibility of using these biomaterials in different maxillofacial surgeries. We conducted an in vivo animal study to evaluate host tissue response to four generations of implanted biomaterial sheets, namely, PLLA, PLLA/PGA, u-HA/PLLA, and u-HA/PLLA/PGA. New bone volume and pertinent biomarkers for bone regeneration, such as Runx2, osteocalcin (OCN), and the inflammatory marker CD68, were analyzed, and the expression of each biomarker was correlated with soft tissues outside the biomaterial and toward the host bone at the end of week 2 and week 10. The use of first-generation biomaterials for maxillofacial osteosynthesis is not advantageous over the use of other updated biomaterials. Second-generation biomaterials degrade faster and can be potentially used in non-stress regions, such as the midface. Third and fourth-generation biomaterials possess bioactive/osteoconductivity improved strength. Application of third-generation biomaterials can be considered panfacially. Fourth-generation biomaterials can be worth considering applying at midface due to the shorter degradation period.

Hematopoietic Stem Cell Numbers Are Regulated at Both Systemic and Local Levels

Since Schofield proposed the niche model in the 1970s, it has been widely believed that the number of hematopoietic stem cells (HSCs) is determined by the niche size. We and others have identified several components of the HSC niche, including Nestin-GFP + mesenchymal stem cells (MSCs). However, the number of defined niche cells vastly exceeds that of HSCs, raising the question about the actual regulatory mechanisms governing HSC numbers. To address this question, we initially established a mathematical model of niche availability and occupancy. The model predicted that an increase in available niches initially leads to a corresponding rise in the number of occupying HSCs, which aligns with the concept of local, niche availability-driven restriction on HSC numbers. Interestingly, our model also suggested that this increase in HSC numbers saturates when the number of available niches reaches a critical level. One potential mechanism for this saturation is that HSC numbers may also be regulated at the systemic level, with total HSC numbers in the body being restricted. To experimentally test this model and hypothesis, we developed a bone transplantation technique that allows us to increase the number of available HSC niches. When a single femur (referred to as a graft) was transplanted per mouse (called a host), we observed the progressive regeneration of bone marrow (BM) stroma and hematopoietic recovery in the grafts. While the number of HSCs (Lin -Sca-1 +c-Kit +CD150 +CD48 -CD34 -) in the grafts was still 41.7 ± 7.60% of that in the host femurs at 5 months after transplantation (n = 8, P = 0.0002), they were comparable at 3 months after transplantation when granulocyte colony-stimulating factor (G-CSF) was administered to hosts at 1 month after transplantation (n = 8, P = 0.660). By utilizing Nestin-GFP transgenic mice and a CD45 congenic system, we found that MSCs in grafts are derived from the graft, while hematopoietic cells are derived from the host. Given that a single femur harbors only 8.3 ± 0.440% of HSCs in the total body (n = 11), 6 femurs were transplanted per mouse, followed by G-CSF administration, to assess the impact of increased HSC niches on total HSC numbers. Strikingly, HSC numbers per host femur and graft femur were lower than those per femur from sham-operated mice (n = 7-48, P = 0.012). Moreover, we found that the sum of HSC numbers in the bone transplantation host and graft bones was equivalent to that in the sham-operated group (n = 7-8, P = 0.364). Total HSC numbers were also maintained when 6 femurs from wild-type (WT) mice were transplanted into Cdh2 (N-Cadherin)-CreER; Cxcl12fl/fl mice, which have an impaired ability to retain HSCs in the BM (n = 8, P = 0.711). These results indicate that the addition of normal niches does not alter total HSC numbers in the body, regardless of whether the endogenous niche is functional, supporting the conclusion that HSC numbers are indeed restricted at the systemic level (Figure 1A). To determine whether HSC numbers are also limited at the local level, as suggested by our mathematical model, we transplanted a single WT femur to Cxcl12fl/fl or Cdh2-CreER; Cxcl12fl/fl mice. We hypothesized that implanting only one femur would have a minimal impact on the total BM, and therefore HSC numbers in the grafts transplanted to Cxcl12fl/fl mice would reach the physiological level. We reasoned that if there is no local restriction on HSC numbers, there would be more HSCs in the WT grafts transplanted to Cdh2-CreER; Cxcl12fl/fl mice compared with those implanted to Cxcl12fl/fl mice. As expected, HSC numbers per WT graft femur in Cxcl12fl/fl mice were comparable to those per femur of sham-operated Cxcl12fl/fl mice (corresponding to the physiological level) (n = 8, P = 0.433). However, CXCL12 deficiency in the host BM did not affect HSC numbers per WT graft femur (n = 8, P = 0.986). Importantly, the sum of HSC numbers in the host body and the graft femur of Cdh2-CreER; Cxcl12fl/fl recipient mice was lower than that of sham-operated Cxcl12fl/fl mice (n = 8, P = 0.003), indicating that total HSC numbers in the former mice are below the systemic limit. Collectively, these data suggest that the failure to increase HSC numbers per WT graft in Cdh2-CreER; Cxcl12fl/fl hosts is, at least in part, due to a local restriction on HSC numbers (Figure 1B). Our study demonstrates that HSC numbers are restricted at both systemic and local levels, thereby rewriting the long-standing model for the regulation of HSC numbers.

Narazaciclib, a Differentiated CDK4/6 Antagonist, Prolongs Cell Cycle Arrest and Metabolomic Reprogramming, Enabling Restoration of Ibrutinib Sensitivity in Btki-Resistant Mantle Cell Lymphoma

Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL. We compared the efficacy and safety profiles of narazaciclib with three health authority-approved CDKi (palbociclib, abemaciclib or ribociclib) in association with covalent (ibrutinib, acalabrutinib) and non-covalent (pirtobrutinib, ARQ-531) BTKi, across a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We integrated RNA sequencing and gene set enrichment analysis (GSEA) coupled to phospho-proteomics and kinase-substrate enrichment analysis (KSEA) to unravel the molecular bases of BTKi-CDKi drug interaction in MCL. CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, RT-PCR and western blot approaches followed by Seahorse-mediated metabolic assays were carried out for validation purposes. Finally, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in three immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft models. We found that narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC 50: 3.61 ± 2.1 µM), regardless of the sensitivity of these to ibrutinib. Although there was no correlation between CDKi sensitivity and direct repression of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response upon narazaciclib treatment. This feature was linked to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second-generation acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTKi-sensitive and BTKi-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome, although similar features were found when substituting narazaciclib with abemaciclib. The integration of transcriptomic and phospho-proteomic synergistic signatures further revealed that narazaciclib-ibrutinib combo modulated signatures related to DNA repair, P53 signaling, and glycolytic activity. Accordingly, in this model, narazaciclib/ibrutinib-exposed cells underwent a metabolic switch from glycolytic to oxidative phosphorylation (OxPhos) phenotype accompanied by intracellular accumulation of mitochondria and increased mtDNA copy number. This switch appeared to be specific to MCL cells rendered refractory to ibrutinib upon activation of alternative NFkB signaling, as it was not found in MCL-BTKmut cells. In vivo, while narazaciclib single agent achieved a moderate TGI in the CAM model, the narazaciclib/ibrutinib combo reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into host bone marrow, with no detectable toxicity. In conclusion, our findings demonstrate that narazaciclib is safe and effective as a single agent in preclinical models of MCL, including BTKi-resistant cases. Its combination with ibrutinib achieved a synergistic tumoricidal effect in vitro and in vivo, accelerating cell cycle blockade and reverting the metabolic reprogramming characterizing MCL refractoriness to BTKi therapy.

Costal Cartilage Graft Repair Osteochondral Defect in a Mouse Model.

Osteochondral defects develop into osteoarthritis without intervention. Costal cartilage can be utilized as an alternative source for repairing osteochondral defect. Our previous clinical study has shown the successful osteochondral repair by costal cartilage graft with integration into host bone bed. In this study, we investigate how cartilaginous graft adapt to osteochondral environment and the mechanism of bone-cartilage interface formation. Costal cartilage grafting was performed in C57BL/6J mice and full-thickness osteochondral defect was made as control. 3D optical profiles and micro-CT were applied to evaluate the reconstruction of articular cartilage surface and subchondral bone as well as gait analysis to evaluate articular function. Histological staining was performed at 2, 4, and 8 weeks after surgery. Moreover, costal cartilage from transgenic mice with fluorescent markers were transplanted into wild-type mice to observe the in vivo changes of costal chondrocytes. At 8 weeks after surgery, 3D optical profiles and micro-CT showed that in the graft group, the articular surface and subchondral bone were well preserved. Gait analysis and International Cartilage Repair Society (ICRS) score evaluation showed a good recovery of joint function and histological repair in the graft group. Safranin O staining showed the gradual integration of graft and host tissue. Costal cartilage from transgenic mice with fluorescent markers showed that donor-derived costal chondrocytes turned into osteocytes in the subchondral area of host femur. Costal cartilage grafting shows both functional and histological repair of osteochondral defect in mice. Graft-derived costal chondrocytes differentiate into osteocytes and contribute to endochondral ossification.

A Novel Screwless Modification of the Khoury Plate Technique using an Innovative Bone Adhesive Formulated from Underwater Biomimetic Marine Proteins.

The Khoury plate technique has been widely utilized with much success over the past 2 decades to facilitate bone augmentations in critically deficient bone areas. Simply, the technique encompasses harvesting autogenous bone plates and utilizing them with external screw fixation. The rigidity of the bone plates enclosing the bony defect on both the buccal and lingual surfaces allows for tension free bone remodeling to occur favoring an ideal bone forming environment. Following, a 4-6 month healing period, a flap is raised, screws may be removed and implant placement then follows. With advancements made in tissue engineering, recently a novel mineral-organic resorbable bone adhesive (MORBA) formulated from underwater biomimetic marine proteins has been utilized for immediate fixation of either bone-to-bone or bone-to-metal. MORBA is a synthetic, injectable, self-setting, and resorbable load-bearing adhesive biomaterial that exhibits over 300 pounds of fixation strength. The unique adhesive properties of MORBA enable it to provide immediate stabilization of dental implants into host bone. The present article describes a novel modification of the Khoury plate technique by utilizing MORBA as a substitute for screws during the fixation of bone plates. A step by step protocol is described where this novel biomaterial was utilized as a substitute to screws favoring a more biological approach to this conventional technique. This novel modification is both surgically easier to conduct, and favors a more biocompatible option that avoids screw fixation and removal.