The enthesis is a tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Given the avascularity of the tendon and the gradual change in tissue architecture and cell phenotype, the enthesis original tissue is often not re-established after chronic injuries, resulting in scar formation. Conservative treatments and surgical approaches are still far from a functional regeneration, whilst tissue engineering based scaffolds have recently showed great potential. In this work, we hypothesised that collagen-based scaffolds that mimic the basic architecture of the enthesis, will be able to spatially direct stem cell differentiation, providing an in vitro platform to study enthesis regeneration. A three-layer sponge composed of a tendon-like layer (collagen type I), a fibrocartilage-like layer (collagen type II) and a bone-like layer (collagen type I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold pore size and structural continuity at the interfaces were assessed by SEM and μ-CT analysis. Bone-marrow derived stem cells (BMSCs) were seeded on the scaffold and cultured in basal and differentiation media (chondrogenic, tenogenic and osteogenic). At day 7 and 21 the scaffolds were stained with Alizarin Red and Alcian Blue; alkaline phosphatase activity (ALP) and calcium and glycosaminoglycans (GAGs) were quantified in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I, III, tenascin and decorin in the scaffolds was evaluated by immunofluorescence staining in order to evaluate tenogenic differentiation of BMSCs. Scaffolds with three distinct but interconnected layers of collagen type I, collagen type II and collagen type I + hydroxyapatite were fabricated, with pore sizes in the range of 100–200 μm. Increased ALP and calcium levels were detected in a localised manner within the bone-like layer when scaffolds were cultured in basal medium (p<0.025 vs the other 2 layers). Similarly, proteoglycans were detected specifically in the fibrocartilage-like layer when scaffolds were cultured in the chondrogenic differentiation medium (p<0.03 vs the other 2 layers). Increased expression of tenogenic markers was observed in the tendon-like layer of scaffolds cultured in tenogenic media (p<0.045 vs the other 2 layers). In conclusion, the different collagen composition of each layer was able to spatially direct BMSC differentiation in a localized manner within the scaffold. Ongoing work is evaluating the synergistic effect between growth factor functionalized within the fibrocartilage and tendon-like layers for improved BMSC differentiation. Overall, these scaffolds hold promising potential in developing novel and more efficient strategy towards enthesis regeneration

This study aimed to characterise the microarchitecture of bone in different species of animal leading to the development of a physiologically relevant 3D printed cellular model of trabecular (Tb) and cortical bone (CB). Using high resolution micro-computed tomography (μ-CT) bone samples from multiple species were scanned and analysed before creating in silico models for 3D printing. Biologically relevant printing materials with physical characteristics similar to that of in vivo bone will be selected and tested for printability. Porcine and murine bone samples were scanned using μ-CT, with a resolution of 4.60 μM for murine and 11 μM for porcine and reconstructed to determine the architectural properties of both Tb and CB independently. A region of interest, 1 mm in height, will be used to generate an in-silico 3D model with dimensions (10 mm. 3. ) and suitable resolution before being translated into printable G code using CAD assisted software. A 1 mm section of each bone was analysed, to determine the differences in the microarchitecture with the intent of setting a benchmark for the developmental 3D in vitro model to be comparable against. In contrast, porcine caudal vertebrae (PCV) have an increased volume due to the size of the bone sample. Interestingly, BV/TR for Tb is similar between species in all samples except murine femur. Murine tibia and PCV have a similar Tb. number and thickness, however different SMI shape and separation. μ-CT scanning and analysis permits tessellation of the 3D output which will lead to the generation of an in silico printable model. Biomaterials are currently under optimisation to allow printability and shape integrity to reflect the morphological and physiological properties of bone

Fracture nonunion is a severe clinical problem for the patient, as well as for the clinician. About 5-20% of fractures does not heal properly after more than six months, with a 19% nonunion rate for tibia, 12% for femur and 13% for humerus, leading to patient morbidity, prolonged hospitalization, and high costs. The standard treatment with iliac crest-derived autologous bone filling the nonunion site may cause pain or hematoma to the patient, as well as major complications such as infection. The application of mesenchymal autologous cells (MSC) to improve bone formation calls for randomized, open, two-arm clinical studies to verify safety and efficacy. The ORTHOUNION * project (ORTHOpedic randomized clinical trial with expanded bone marrow MSC and bioceramics versus autograft in long bone nonUNIONs) is a multicentric, open, randomized, comparative phase II clinical trial, approved in the framework of the H2020 funding programme, under the coordination of Enrique Gòmez Barrena of the Hospital La Paz (Madrid, Spain). Starting from January 2017, patients with nonunion of femur, tibia or humerus have been actively enrolled in Spain, France, Germany, and Italy. The study protocol encompasses two experimental arms, i.e., autologous bone marrow-derived mesenchymal cells after expansion (‘high dose’ or ‘low dose’ MSC) combined to ceramic granules (MBCP™, Biomatlante), and iliac crest-derived autologous trabecular bone (ICAG) as active comparator arm, with a 2-year follow-up after surgery. Despite the COVID 19 pandemic with several lockdown periods in the four countries, the trial was continued, leading to 42 patients treated out of 51 included, with 11 receiving the bone graft (G1 arm), 15 the ‘high dose’ MSC (200x10. 6. , G2a arm) and 16 the ‘low dose’ MSC (100x10. 6. , G2b arm). The Rizzoli Orthopaedic Institute has functioned as coordinator of the Italian clinical centres (Bologna, Milano, Brescia) and the Biomedical Science and Technologies and Nanobiotechnology Lab of the RIT Dept. has enrolled six patients with the collaboration of the Rizzoli’ 3rd Orthopaedic and Traumatological Clinic prevalently Oncologic. Moreover, the IOR Lab has collected and analysed the blood samples from all the patients treated to monitor the changes of the bone turnover markers following the surgical treatment with G1, G2a or G2b protocols. The clinical and biochemical results of the study, still under evaluation, are presented. * ORTHOUNION Horizon 2020 GA 733288

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients

Femoroacetabular impingement (FAI) results from a morphological deformity of the hip and is associated with osteoarthritis (OA). Increased bone mineral density (BMD) is observed in the antero-superior acetabulum rim where impingement occurs. It is hypothesized that the repeated abnormal contact leads to damage of the cartilage layer, but could also cause a bone remodelling response according to Wolff's Law. Thus the goal of this study was to assess the relationship between bone metabolic activity measured by PET and BMD measured in CT scans. Five participants with asymptomatic cam deformity, three patients with uni-lateral symptomatic cam FAI and three healthy controls were scanned in a 3T PET-MRI scanner following injection with [18F]NaF. Bone remodelling activity was quantified with Standard Uptake Values (SUVs). SUVmax was analyzed in the antero-superior acetabular rim, femoral head and head-neck junction. In these same regions, BMD was calculated from CT scans using the calibration phantom included in the scan. The relationship between SUVmax and BMD from corresponding regions was assessed using the coefficient of determination (R. 2. ) from linear regression. High bone activity was seen in the cam deformity and acetabular rim. SUVmax was negatively correlated with BMD in the antero-superior region of the acetabulum (R. 2. =0.30, p=0.08). SUVmax was positively correlated with BMD in the antero-superior head-neck junction of the femur (R. 2. =0.359, p=0.067). Correlations were weak in other regions. Elevated bone turnover was seen in patients with a cam deformity but the relationship to BMD was moderate. This study demonstrates a pathomechanism of hip degeneration associated with FAI deformities, consistent with Wolff's law and the proposed mechanical cause of hip degeneration in FAI. [18F]-NaF PET SUV may be a biomarker of degeneration, especially in early stages of degeneration, when joint preservation surgery is likely to be the most successful

Our previous research has demonstrated that minor adjustments to in vitro cellular aggregation parameters, i.e. alterations to aggregate size, can influence temporal and spatial mineral depositions within maturing bone cell nodules. What remains unclear, however, is how aggregate size might affect mineralisation within said nodules over long-term in vivo culture. In this study, we used an osteoblast cell line, MLO-A5, and a primary cell culture, mesenchymal stem cells (MSC), to compare small (approximately 80 µm) with large (approximately 220 µm) cellular aggregates for potential bone nodule development after 8 weeks of culturing in a mouse model (n = 4 each group). In total, 30 chambers were implanted into the intra-peritoneal cavity of 20 male, immunocompromised mice (MF1-Nu/Nu, 4 – 5 weeks old). Nine small or three large aggregates were used per chamber. Neoveil mesh was seeded directly with 2 × 10. 3. cells for monolayer control. At 8 weeks, the animals were euthanised and chambers fixed with formalin. Aggregate integrity and extracellular material growth were assessed via light microscopy and the potential mineralisation was assessed via micro-CT. Many large aggregates appeared to disintegrate, whilst the small aggregates maintained their form and produced additional extracellular material with increased sizes. Both MLO-A5 cells and MSC cells saw similar results. Interestingly, however, the MSCs were also seen to produce a significantly higher volume of dense material compared to the MLO-A5 cells from micro-CT analysis. Overall, a critical cell aggregate size appeared to exist balancing optimal tissue growth with oxygen diffusion, and cell source may influence differentiation pathway despite similar experimental parameters. The MSCs, for example, were likely producing bone via the endochondral ossification pathway, whilst the matured bone cells, MLO-A5 cells, were likely producing bone via the intramembranous ossification pathway

Bone infections due to fractures or implants are a big medical problem. In experimental medicine, many experimental models have been created on different animal species to simulate the disease condition and to do experience treatments. The aim of this paper was to present an antibacterial efficacy of using a bone allograft developed according to the Marburg system of bone bank on a model of chronic osteomyelitis induced in rabbits. In research was used 54 rabbits. Osteomyelitis was induced in rabbits by a human strain of St. aureus ATCC 43300, in the rabbit femur. There have been created 3 groups of animals. In 1. st. group used antibiotic impregnated biodegradable material “PerOssal”. In 2. nd. group used antibiotic impregnated whole bone allograft. In 3. rd. group used antibiotic impregnated perforated bone allograft. Evaluation of installation and evolution of the disease was done by microbiological. A separate study of microbiological data is presented here. This study showed, in the 1. st. and 3. rd. groups there is a persistent decrease in CFU by 14 knocks to 120.4 in the 1. st. group and to 3.5 in the 3. rd. group, and in the 2. nd. group, on the contrary, there is an increase in CFU to 237.33. This shows the lack of effectiveness of using a whole bone allograft. The results showed, after 7 days there was no statistically significant difference between the groups. After 14 days the perforated bone allograft impregnated with antibiotic was better than the biodegradable material “PerOssal”

Autografts containing bone marrow (BM) are current gold standard in the treatment of critical size bone defects, delayed union and bone nonunion defects. Although reaching unprecedented healing rates in bone reconstruction, the mode of action and cell-cell interactions of bone marrow mononuclear cell (BM-MNC) populations have not yet been described. BM-MNCs consist of a heterogeneous mixture of hematopoetic and non-hematopoetic lineage fractions. Cell culture in a 3D environment is necessary to reflect on the complex mix of these adherend and non-adherend cells in a physiologically relevant context. Therefore, the main aim of this approach was to establish conditions for a stable 3D BM-MNC culture to assess cellular responses on fracture healing strategies. BM samples were obtained from residual material after surgery with positive ethical vote and informed consent of the patients. BM-MNCs were isolated by density gradient centrifugation, and cellular composition was determined by flow cytometry to obtain unbiased data sets on contained cell populations. Collagen from rat tail and human fibrin was used to facilitate a 3D culture environment for the BM-MNCs over a period of three days. Effects on cellular composition that could improve the regenerative potential of BM-MNCs within the BM autograft were assessed using flow cytometry. Cell-cell-interactions were visualized using confocal microscopy over a period of 24 hours. Cell localization and interaction partners were characterized using immunofluorescence labeled paraffin sectioning. Main BM-MNC populations like Monocytes, Macrophages, T cells and endothelial progenitor cells were determined and could be conserved in 3D culture over a period of three days. The 3D cultures will be further treated with already clinically available reagents that lead to effects even within a short-term exposure to stimulate angiogenic, osteogenic or immunomodulatory properties. These measures will help to ease the translation from “bench to bedside” into an intraoperative protocol in the end

Introduction and Objective. Management of bone loss associated with bone contamination or infection represents a double biological and clinical challenge frequent in traumatology. The advent of new biomaterials can allow a different approach in the treatment of bone gap. The purpose of this study was to evaluate the prophylactic and therapeutic effectiveness of addition of a new absorbable bone substitute (BS) eluting different antibiotics in reconstruction of bone defects after infections and fractures with soft tissue damage. Materials and Methods. We conducted a review of patients with contaminated or infected bone defects treated using a new biomaterial, a porous composite of collagen matrices and Beta tricalcium phosphate (β TCP), able to provide a long-term release of different antibiotics. We have included treatment of osteomyelitis and osteosynthesis of exposed fracture (Gustilo Anderson 1–3b) or fractures with soft tissue damage and high risk of contamination. Surgical technique included debridement filling bone defect with BS eluting antibiotics, osteosynthesis (plate, nail, external fixator, kirschner wire), soft tissue coverage, and systemic antibiotic therapy. Radiographic and clinical data including complications (wound dehiscence, superficial or deep infection, osteomyelitis) were collected. Results. We treated 25 patients (21 male, 4 female) with mean age 47 yrs. (range 21–83). The locations treated (for incidence) was: 9 femurs (7 plates, 2 nail), 7 calcanei (one bilateral), 3 tibias, 2 forearms, 2 metatarsi, 2 hands, 1 elbow. 6 patients had large bone loss. 7 patients had bone infections (4 were Cierny Madern 4); 8 patients had osteosynthesis of exposed fractures Gustilo Anderson 1–3b (9 plate, one bilateral calcaneus). 8 patients had treatment for pseudoarthrosis of exposed fractures (6 femurs, 1 forearm, 1 metatarsus) and 3 patients a prophylactic treatment for calcaneal fractures with soft tissue damage. 4 deep infection were treated with multiple surgical debridement and new filling bone defect with BS eluting antibiotic with infection eradication. We have used a combination of vancomycin and gentamicin on 15 cases, vancomycin alone on 4 cases, combination of vancomycin and amikacin on 1 case and amikacin and Linezolid in a targeted multi drug resistance. At final follow-up functional outcome was good in all cases with bone healing. Conclusions. Extensive debridement is a fundamental requisite for eradication of bone infections and contamination. Filling of the bone void with loaded bio-composite eluting diversifiable local antibiotics with synergistic anti-biofilm activity is desirable. Treatment of this bone defects are advantaged when combining his reconstruction with BS and the possibility of release high antibiotic concentration at least for 10 days. This is an important complementing prophylactic and therapeutic antimicrobial option with adjuvant role to systemic therapy that enlarges the success rate

Introduction and Objective. In recent years, along with the extending longevity of patients and the increase in their functional demands, the number of annually performed RSA and the incidence of complications are also increasing. When a complication occurs, the patient often needs multiple surgeries to restore the function of the upper limb. Revision implants are directly responsible for the critical reduction of the bone stock, especially in the shoulder. The purpose of this paper is to report the use of allograft bone to restore the bone stock of the glenoid in the treatment of an aseptic glenoid component loosening after a reverse shoulder arthroplasty (RSA). Materials and Methods. An 86-years-old man came to our attention for aseptic glenoid component loosening after RSA. Plain radiographs showed a complete dislocation of the glenoid component with 2 broken screws in the neck of glenoid. CT scans confirmed the severe reduction of the glenoid bone stock and critical bone resorption and were used for the preoperative planning. To our opinion, given the critical bone defect, the only viable option was revision surgery with restoration of bone stock. We planned to use a bone graft harvested from distal bone bank femur as component augmentation. During the revision procedure the baseplate with a long central peg was implanted “on table” on the allograft and an appropriate osteotomy was made to customize the allograft on the glenoid defect according to the CT-based preoperative planning. The Bio-component was implanted with stable screws fixation on residual scapula. We decided not to replace the humeral component since it was stable and showed no signs of mobilization. Results. The new bio-implant was stable, and the patient gained a complete functional recovery of the shoulder. The scheduled radiological assessments up to 12 months showed no signs of bone resorption or mobilization of the glenoid component. Conclusions. The use of bone allograft in revision surgery after a RSA is a versatile and effective technique to treat severe glenoid bone loss and to improve the global stability of the implant. Furthermore, it represents a viable alternative to autologous graft since it requires shorter operative times and reduces graft site complications. There are very few data available regarding the use of allografts and, although the first studies are encouraging, further investigation is needed to determine the biological capabilities of the transplant and its validity in complex revisions after RSA

Introduction and Objective. Calcium phosphates are among the most commonly used bone graft substitute materials. Compositions containing predominantly monetite (∼84.7%) with smaller additions of beta-tricalcium phosphate (β-TCP; ∼8.3%) and calcium pyrophosphate (Ca-PP; ∼6.8%) have previously been demonstrated to exhibit osteoinductive properties. Such a multi-component calcium phosphate bioceramic was fashioned in the form of hollowed-out, dome-shaped devices (15 mm diameter, 4 mm height), each reinforced with a 3D printed Ti6Al4V ELI frame. With the aim to induce bone formation beyond the skeletal envelope, these devices were investigated in vivo using a sheep (Ovis aries) occipital bone model. Materials and Methods. The bioceramic composition was prepared from a mixture of β-TCP/dicalcium pyrophosphate and monocalcium phosphate monohydrate powders mixed with glycerol. The Ti6Al4V ELI frame was positioned into a dome-shaped mould and bioceramic paste was poured over the frame and allowed to set, in sterile water, prior to removal from the mould. In adult female sheep (n=7), the devices were positioned directly over the bone and stabilised using self-drilling screws. After 52 weeks, the devices were retrieved, resin embedded, and used for X-ray micro-computed tomography (micro-CT), histology, backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), micro-Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Results. The bioceramic composition (Ca/P: ∼0.85 at. %) transforms to carbonated apatite (Ca/P: ∼1.2 at. %, Mg/Ca: ∼0.03 at. %), in vivo, largely at the expense of monetite and Ca-PP whereas β-TCP remains detectable. Discrete particles of Ca-PP are identified by correlative BSE-SEM and micro-Raman spectroscopy. Together with chemical transformation, physical degradation is evident within the bulk of the bioceramic. Beyond the confines of the skeletal envelope, de novo bone occupies ∼53–84% (∼73 ± 11%; mean ± standard deviation) of the hollowed-out space. Low porosity and the arrangement of remodelled bone into a concentric lamellar pattern is indicative of cortical-like structure. Such areas are typically surrounded by yet unremodelled, and microstructurally disordered, woven bone that stains intensely with blue cationic dyes, owing to relatively higher acid phosphate content. This pattern indicates a recurring sequence of woven bone formation followed by remodelling. Bone formation is also visible within the bioceramic. Recently remodelled and areas of ongoing remodelling are identified by relatively lower mineral density than the surrounding woven bone. Dendritic extensions of osteocytes appear to extend into the bioceramic surface. Both micro-Raman spectroscopy and FTIR reveal little, if any, detectable difference between the mineral and organic phases of the extracellular matrix, between de novo and native bone. Conclusions. The bioceramic composition undergoes physical degradation, but remains largely intact by 52 weeks in vivo, and only partially transforms to carbonated apatite. In addition to very high bone volume within the hollowed-out bioceramic device, the overall composition and microstructure of de novo bone are similar to native bone. Notably, the mineral phase of bone in response to, and in direct contact with the β-TCP, monetite, and Ca-PP, remains exclusively carbonated apatite

While knee osteoarthritis (OA) is now recognized as a complex disease affecting the whole joint, not just the cartilages, there remains a paucity of data regarding the interactions between knee components. One relationship of particular interest is between the spatial variations in cartilage thickness (CTh) and subchondral bone mineral density (BMD). Indeed, bone and cartilage are two mechanosensitive tissues that interact as a functional unit and there is evidence of a biomechanical coupling between both tissues. Particularly, a recent in vivo study has shown a positive relationship in non-OA knees with thicker cartilage where bone is denser, and an alteration of this relationship in OA knees. These observations support the concept of an osteochondral unit and warrant additional research to assess the influence of bone depth. Therefore, this study aimed to characterize the relationship between spatial variations in CTh and BMD measured at various depths below the bone surface. CT-arthrography of 20 non-OA tibias and 20 severe medial-compartment OA tibias were segmented to build 3D mesh models of the bones and cartilages. Each individual tibia model was registered to a reference tibia, allowing to calculate BMD maps at 1, 3, 5 and 10mm below the bone-cartilage interface in the medial compartment. Pearson correlations between CTh maps and the four BMD maps were then calculated for each knee. Lastly, differences in correlation coefficients between successive bone layers were assessed using Wilcoxon signed-rank tests. In both OA and non-OA tibias, the correlation coefficients were higher with the BMD measured in the 1mm layer, and followed a pattern of statistically significant decrease with bone layers of increasing depth (p < 0.021). In non-OA tibias, the median relationship was positive with a strong effect size in the 1, 3 and 5mm layers, while in OA tibias the median relationship was positive only in the 1mm layer and with a medium effect size. In the OA tibias, the median relationship was negative with a weak effect size in the 3 and 5mm layers, and it was negative with a medium effect size in the 10mm layer. In conclusion, the results of the present study support the value of considering bone and cartilage as a unit, and more generally support OA pathophysiology models based on relationships among knee properties

Abstract. Purpose. It is becoming apparent that mesenchymal stem cells (MSCs) do not directly contribute to mesenchymal tissue regeneration. Pre-clinical attempts to repair large bone defects in big animal models have been hampered by poor MSCs survival after implantation which impedes their direct or indirect effects. Based on previous work, we hypothesized that a venous axial vascularization of the scaffold supporting MSCs or their combination with fresh bone marrow (BM) aspirate would improve their in vivo survival. Methods. Cross-shape profile tubular microporous monetite implants (12mm long, 5mm large) as two longitudinal halves were produced by 3D powder printing. They were implanted around the femoral veins of Wistar rats and loaded with 1mL of BM aspirate either alone or supplemented by 10. 7. MSCs. This was compared with BM-free scaffolds loaded only with 10. 7. MSCs. After 8 weeks bone formation were investigated by micro-CT, scanning electron microscopy, histology and immunohistochemistry. Results. Little bone formation was observed within the scaffold when it was only loaded with MSCs surprisingly. Coupling MSCs, autologous BM and venous perfusion of the scaffold led to a higher volume of new bone than BM alone suggesting that MSCs augmented the bone formation capacity of BM aspirate or enhanced its survival post implantation. Conclusion. Subcutaneous bone formation within 3D-printed implant that mixed of BM with or without MSCs was successfully achieved for the first time by venous perfusion. The inability of MSCs to form differentiated tissues by their own was confirmed in this study; however, contact between MSCs and BM cells and MSCs paracrine secretome (e.g., cytokines, chemokines, extracellular vesicles) may have induced immunomodulatory effects (e.g., macrophages polarization, Treg cells) that triggered bone formation. This approach, if translatable to large animal models, offers immediate clinical value as well as an insight into the role of immune system in tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported: I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic controls. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Our studies suggest that aged BMSCs regenerative activity can be enhanced by culture on hiPSC-engineered ECM

A spine compression fracture is a very common form of fracture in elderly with osteoporosis. Injection of polymethyl methacrylate (PMMA) to fracture sites is a minimally invasive surgical treatment, but PMMA has considerable clinical risks. We develop a novel type thermoplastic injectable bone substitute contains the proprietary composites of synthetic ceramic bone substitute and absorbable thermoplastic polymer. We used thermoplastic biocompatible polymers Polycaproactone (PCL) to encapsulate calcium-based bone substitutes hydroxyapatite (Ca10(PO4)6(OH)2, HA) and tricalcium phosphate (TCP) to form a biodegradable injectable bone composite material. The space occupation ration PCL:HA/TCP is 1:9. After heating process, it can be injected to fracture site by specific instrument and then self-setting to immediate reinforce the vertebral body. The thermoplastic injection bone substitute can obtain good injection properties after being heated by a heater at 90˚C for three minutes, and has good anti-washout property when injected into normal saline at 37˚C. After three minutes, solidification is achieved. Mechanical properties were assessed using the material compression test system and the mechanical support close to the vertebral spongy bone. In vitro cytotoxicity MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed and no cell cytotoxicity was observed. In vivo study with three New Zealand rabbits was performed, well bone growth into bone substitute was observed and can maintain good mechanical support after three months implantation. The novel type thermoplastic injection bone substitute can achieve (a) adequate injectability and viscosity without the risk of cement leakage; (b) adequate mechanical strength for immediate reinforcement and prevent adjacent fracture; (c) adequate porosity for new bone ingrowth; (e) biodegradability. It could be developed as a new option for treating vertebral compression fractures

Abstract. Objectives. Young patients receiving metallic bone implants after surgical resection of bone cancer require implants that last into adulthood, and ideally life-long. Porous implants with similar stiffness to bone can promote bone ingrowth and thus beneficial clinical outcomes. A mechanical remodelling stimulus, strain energy density (SED), is thought to be the primary control variable of the process of bone growth into porous implants. The sequential process of bone growth needs to be taken into account to develop an accurate and validated bone remodelling algorithm, which can be employed to improve porous implant design and achieve better clinical outcomes. Methods. A bone remodelling algorithm was developed, incorporating the concept of bone connectivity (sequential growth of bone from existing bone) to make the algorithm more physiologically relevant. The algorithm includes adaptive elastic modulus based on apparent bone density, using a node-based model to simulate local remodelling variations while alleviating numerical checkerboard problems. Strain energy density (SED) incorporating stress and strain effects in all directions was used as the primary stimulus for bone remodelling. The simulations were developed to run in MATLAB interfacing with the commercial FEA software ABAQUS and Python. The algorithm was applied to predict bone ingrowth into a porous implant for comparison against data from a sheep model. Results. The accuracy of the predicted bone remodelling was verified for standard loading cases (bending, torsion) against analytical calculations. Good convergence was achieved. The algorithm predicted good bone remodelling and growth into the investigated porous implant. Using the standard algorithm without connectivity, bone started to remodel at locations unconnected to any bone, which is physiologically implausible. The implementation of bone connectivity ensures the gradual process of bone growth into the implant pores from the sides. The bone connectivity algorithm predicted that the full remodelling required more time (approximately 50% longer), which should be considered when developing post-surgical rehabilitation strategies for patients. Both algorithms with and without bone connectivity implementation converged to same final stiffness (less than 0.01% difference). Almost all nodes reached the same density value, with only a limited number of nodes (less than 1%) in transition areas with a strong density gradient having noticeable differences. Conclusions. An improved bone remodelling algorithm based on strain energy density that modelled the sequential process of bone growth has been developed and tested. For a porous metallic bone implant the same final bone density distribution as for the original adaptive elasticity theory was predicted, with a slower and more fidelic process of growth from existing surrounding bone into the porous implant. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Bone regeneration is an area of acute medical need, but its clinical success is hampered by the need to ensure rapid vascularization of osteogenic grafts. Vascular Endothelial Growth Factor (VEGF) is the master regulator of vascular growth and during bone development angiogenesis and osteogenesis are physiologically coupled through so-called angiocrine factors produced by blood vessels. However, how to exploit this process for therapeutic bone regeneration remains a challenge (1). Here we will describe recent work aiming at understanding the cross-talk between vascular growth and osteogenesis under conditions relevant for therapeutic bone regeneration. To this end we take advantage of a unique platform to generate controlled signalling microenvironments, by the covalent decoration of fibrin matrices with tunable doses and combinations of engineered growth factors. The combination of human osteoprogenitors and hydroxyapatite in these engineered fibrin matrices provides a controlled model to investigate how specific molecular signals regulate vascular invasion and bone formation in vivo. In particular, we found that:. 1). Controlling the distribution of VEGF protein in the microenvironment is key to recapitulate its physiologic function to couple angiogenesis and osteogenesis (2);. 2). Such coupling is exquisitely dependent on VEGF dose and on a delicate equilibrium between opposing effects. A narrow range of VEGF doses specifically activates Notch1 signaling in invading blood vessels, inducing a pro-osteogenic functional state called Type H endothelium, that promotes differentiation of surrounding mesenchymal progenitors. However, lower doses are ineffective and higher ones paradoxically inhibit both vascular invasion and bone formation (Figure 1) (3);. 3). Semaphorin3a (Sema3a) acts as a novel pro-osteogenic angiocrine factor downstream of VEGF and it mediates VEGF dose-dependent effects on both vascular invasion and osteogenic progenitor stimulation. In conclusion, vascularization of osteogenic grafts is not simply necessary in order to enable progenitor survival. Rather, blood vessels can actively stimulate bone regeneration in engineered grafts through specific molecular signals that can be harnessed for therapeutic purposes. Acknowledgements: This work was supported in part by the European Union Horizon 2020 Program (Grant agreement 874790 – cmRNAbone). For any figures and tables, please contact the authors directly

The aim of the ongoing projects was to demonstrate the efficacy of autologous bone marrow derived stem cells (MSC) combined with biomaterial to induced new bone formation in a randomized multicenter controlled clinical trial. Patients with a need for bone reconstruction of residual edentulous ridges in both the mandible and maxilla due to bone defects with a vertical loss of alveolar bone volume and/or knife edge ridges (≤ than 4,5 mm) unable to provide adequate primary stabilization for dental implants were included in the clinical study. Autologous bone marrow MSC were expanded, loaded on BCP and used to augment the alveolar ridges. After five months bone biopsies were harvested at the implant position site and implants were installed in the regenerated bone. The implants were loaded after 8–12 weeks. Safety, efficacy, quality of life and success/survival were assessed. Five clinical centers, 4 different countries participated. Bone grafts harvested from the ramus of the mandibles were used as control in the projects

Primary bone tumors are rare, complex and highly heterogeneous. Its diagnostic and treatment are a challenge for the multidisciplinary team. Developments on tumor biomarkers, immunohistochemistry, histology, molecular, bioinformatics, and genetics are fundamental for an early diagnosis and identification of prognostic factors. The personalized medicine allows an effective patient tailored treatment. The bone biopsy is essential for diagnosis. Treatment may include systemic therapy and local therapy. Frequently, a limb salvage surgery includes wide resection and reconstruction with endoprosthesis, biological or composites. The risk for local recurrence and distant metastases depends on the primary tumor and treatment response. Cancer patients are living longer and bone metastases are increasing. Bone is the third most frequently location for distant lesions. Bone metastases are associated to pain, pathological fractures, functional impairment, and neurological deficits. It impacts survival and patient quality of life. The treatment of metastatic disease is a challenge due to its complexity and heterogeneity, vascularization, reduced size and limited access. It requires a multidisciplinary treatment and depending on different factors it is palliative or curative-like treatment. For multiple bone metastases it is important to relief pain and increases function in order to provide the best quality of life and expect to prolong survival. Advances in nanotechnology, bioinformatics, and genomics, will increase biomarkers for early detection, prognosis, and targeted treatment effectiveness. We are taking the leap forward in precision medicine and personalized care

There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet. Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity. In vitro, an increased gene expression of extracellular matrix proteins as collagen II or aggrecan even under inflammatory conditions was observed under Mg-impact. In vivo, µCT evaluation revealed twice-elevated values for bone volume in femoral condyles with Mg-cylinders compared to controls while density remained unchanged. Cartilage showed no significant differences between the groups. Mg- and WE-samples showed significantly lower levels of CD271+ cells in the cartilage and bone of the operated joints than in non-operated joints, which was not the case in the Drilling-group. Furthermore, bone in operated knees of Drilling-group showed a strong trend to an increase in CD271+ cells compared to both Cylinder-groups. Counting of CD271+ vessels revealed that this difference was attributable to a higher amount of these vessels. The in vitro results indicate a potential cartilage regenerative activity of the degradable Mg-based material. While so far there was no positive effect on the cartilage itself in vivo, implantation of Mg-cylinders seemed to reduce pain-mediating vessels. Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 404534760). We thank Björn Wiese for production of the cylinders