Introduction. Herein, a tri-layered core-shell microfibrous scaffold with layer-specific growth factors (GFs) release is developed using coaxial electrohydrodynamic (EHD) printing for in situ cell recruitment and differentiation to facilitate gradient enthesis tissue repair. Our findings suggest that the microfibrous scaffolds with layer-specific GFs release may offer a promising clinical solution for enthesis regeneration. Method. Utilizing coaxial electrohydrodynamic (EHD) printing, we engineered tri-layered core-shell microfibrous scaffolds, each layer tailored with specific growth factors (GFs) for targeted enthesis tissue repair. This configuration aims to sequentially guide cell migration and differentiation, mirroring the natural enthesis’ gradient structure. SDF-1 was strategically loaded into the shell, while bFGF, TGF-β, and BMP-2 were encapsulated in the core, each selected for their roles in stimulating the regeneration of corresponding enthesis tissue layers. Result. The coaxial EHD-printed microfibrous scaffolds demonstrated a core-shell fiber width of 24.3 ± 6.3 μm, supporting distinct tenogenic, chondrogenic, and osteogenic layers with pore sizes of 81.5 ± 4.6 μm, 173.3 ± 6.9 μm, and 388.9 ± 6.9 μm, respectively. This structure facilitated a targeted and effective release of growth factors, optimizing stem cell recruitment and differentiation. In vivo assessments demonstrated that the scaffolds significantly enhanced biomechanical properties and facilitated the formation of gradient enthesis structures, with improved biomechanical strength approximately 2-3 times that of control groups. These results highlight the scaffold's capability to mimic the native enthesis structure, encouraging a conducive environment for cell-mediated repair and regeneration. Conclusion. The integration of layer-specific growth factors not only fostered a conducive environment for tissue regeneration but also exemplified a leap in the design of scaffolds that closely mimic the native tendon-to-bone interface. The findings illuminate the scaffold's capacity to direct cellular behavior and tissue formation, heralding a new era in regenerative strategies and offering a promising avenue for clinical translation in the treatment of rotator cuff injuries

Introduction. Immunomodulation represents a novel strategy to improve bone healing in combination with low doses of bone morphogenetic growth factors like BMP-2. This study aims to investigate the effect and timing of monoclonal anti-IL-1ß antibody administration with 1μg BMP-2 on bone healing over 14 weeks in a rat femur segmental defect model. Method. 2 mm femoral defects were created in 22-27 weeks-old female Fischer F344 rats, internally fixed with a plate (animal license: GR/19/2022) using established protocols for analgesia and anesthesia. Animals (n=4/group) received either a collagen sponge, a collagen sponge+1μg BMP-2 (InductOs, Medtronic) or a collagen sponge+1μg BMP-2 with a monoclonal anti-IL-1ß antibody (BioXCell, 10 mg/ml), administered intravenously under anesthesia every third day until day 15, from day 0 or 3. In vivo micro-CT was performed after surgery and at 2, 3, 4, 6, 8, 10 and 14-weeks post-OP. Mechanical properties of the operated femurs were assessed by 4-point bending (Instron5866) and compared to contralateral femurs (one-way ANOVA, GraphPad Prism8). Histopathological analysis was performed semi-quantitatively on Giemsa-Eosin-stained sections (Olympus BX63) using a six-grade severity grading scale. Result. Operated femurs with BMP-2 reached an average stiffness of 91±37% of contralateral femurs, femurs in IL-1ß groups 105±11% (day 0) and 111±12% (day 3). Administration of anti-IL-1ß+1μg BMP-2 led to faster cortical bridging (3/4 femurs bridged by week 4 for day 0, 4/4 for day 3) than 1μg BMP-2 alone (0/4 by week 4). Micro-CT results confirmed histopathological evaluation, as collagen sponge alone led to non-union, complete bicortical bridging was observed for 3/4 femurs in the BMP-2 group and for 4/4 femurs in the IL-1β groups after 14 weeks. Conclusion. Anti-IL-1ß had a beneficial effect on late fracture healing with faster cortical bridging and new bone formation than 1μg BMP-2 alone. Acknowledgments. AO foundation. We thank Andrea Furter, Alisa Hangartner and Thomas Krüger for technical support

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Aims. To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration. Methods. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm. 2. , 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens. Results. In the histopathological analysis, the macro-morphological grading scale showed a significant increase, while the histological score and cartilage repair scale of ESWT exhibited a significant decrease compared to OCD at the 8- and 12-week timepoints. At the 12-week follow-up, ESWT exhibited a significant improvement in the volume of damaged bone compared to OCD. Furthermore, immunohistochemistry analysis revealed a significant decrease in type I collagen and a significant increase in type II collagen within the newly formed hyaline cartilage following ESWT, compared to OCD. Finally, SRY-box transcription factor 9 (SOX9), aggrecan, and TGF-β, BMP-2, -3, -4, -5, and -7 were significantly higher in ESWT than in OCD at 12 weeks. Conclusion. ESWT promoted the effect of TGF-β/BMPs, thereby modulating the production of extracellular matrix proteins and transcription factor involved in the regeneration of articular cartilage and subchondral bone in an OCD rat model. Cite this article: Bone Joint Res 2024;13(7):342–352

There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture. To assess the optimal concentration of MSCs for promoting fracture healing in a rat model. Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 자 post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-β1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-β1 and VEGF] was evaluated using real-time polymerase chain reaction. Micro-CT showed that BV and PBV was significantly increased in groups M and H compared to that in group C at 6 wk post-fracture (P = 0.040, P = 0.009; P = 0.004, P = 0.001, respectively). Significantly more cartilaginous tissue and immature bone were formed in groups M and H than in group C at 2 and 6 wk post-fracture (P = 0.018, P = 0.010; P = 0.032, P = 0.050, respectively). At 2 wk post fracture, SDF-1, TGF-β1 and VEGF expression were significantly higher in groups M and H than in group L (P = 0.031, P = 0.014; P < 0.001, P < 0.001; P = 0.025, P < 0.001, respectively). BMP-2 and VEGF expression were significantly higher in groups M and H than in group C at 6 wk postfracture (P = 0.037, P = 0.038; P = 0.021, P = 0.010). Compared to group L, TGF-β1 expression was significantly higher in groups H (P = 0.016). There were no significant differences in expression levels of chemokines related to MSC migration, angiogenesis and cytokines associated with osteogenesis between M and H groups at 2 and 6 wk post-fracture. The administration of at least 5.0 × 106 MSCs was optimal to promote fracture healing in a rat model of long bone fractures

Although bone morphogenetic protein 2 (BMP-2) has been FDA-approved for spinal fusion for decades, its disadvantages of promoting osteoclast-based bone resorption and suboptimal carrier (absorbable collagen sponge) leading to premature release of the protein limit its clinical applications. Our recent study showed an excellent effect on bone regeneration when BMP-2 and zoledronic acid (ZA) were co-delivered based on a calcium sulphate/hydroxyapatite (CaS/HA) scaffold in a rat critical-size femoral defect model. Therefore, the aim of this study was to evaluate whether local application of BMP-2 and ZA released from a CaS/HA scaffold is favorable for spinal fusion. We hypothesized that CaS/HA mediated controlled co-delivery of rhBMP-2 and ZA could show an improved effect in spinal fusion over BMP-2 alone. 120, 8-week-old male Wistar rats (protocol no. 25-5131/474/38) were randomly divided into six groups in this study (CaS/HA, CaS/HA + BMP-2, CaS/HA + systemic ZA, CaS/HA + local ZA, CaS/HA + BMP-2 + systemic ZA, CaS/HA + BMP-2 + local ZA). A posterolateral spinal fusion at L4 to L5 was performed bilaterally by implanting group-dependent scaffolds. At 3 weeks and 6 weeks, 10 animals per group were euthanized for µCT, histological staining, or mechanical testing. µCT and histological results showed that the CaS/HA + BMP-2 + local ZA group significantly promoted bone regeneration than other treated groups. Biomechanical testing showed breaking force in CaS/HA + BMP + local ZA group was significantly higher than other groups at 6 weeks. In conclusion, the CaS/HA-based biomaterial functionalized with bioactive molecules rhBMP-2 and ZA enhanced bone formation and concomitant spinal fusion outcome. Acknowledgements: Many thanks to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology)

To design slow resorption patient-specific bone graft whose properties of bone regeneration are increased by its geometry and composition and to assess it in in-vitro and in-vivo models. A graft composed by hydroxyapatite (HA) and β-TCP was designed as a cylinder with 3D gyroid porosities and 7 mm medullary space based on swine's anatomy. It was produced using a stereolithography 3D-printing machine (V6000, Prodways). Sterile bone grafts impregnated with or without a 10µg/mL porcine BMP-2 (pBMP-2) solution were implanted into porcine femurs in a bone loss model. Bone defect was bi-weekly evaluated by X-ray during 3 months. After sacrifice, microscanner and non-decalcified histology analysis were conducted on biopsies. Finally, osteoblasts were cultured inside the bone graft or in monolayer underneath the bone graft. Cell viability, proliferation, and gene expression were assessed after 7 and 14 days of cell culture (n=3 patients). 3D scaffolds were successfully manufactured with a composition of 80% HA and 20% β-TCP ±5% with indentation compressive strength of 4.14 MPa and bending strength of 11.8MPa. In vivo study showed that bone regeneration was highly improved in presence of pBMP-2. Micro-CT shows a filling of the gyroid sinuses of the implant (Figure 1). In vitro, the presence of BMP2 did not influence the viability of the osteoblasts and the mortality remained below 3%. After 7 days, the presence of BMP2 in the scaffold significantly increased by 85 and 65% the COL1A1 expression and by 8 and 33-fold the TNAP expression by osteoblasts in the monolayer or in the scaffold, respectively. This BMP2 effect was transient in monolayer and did not modify gene expression at day 14. BMP2-impregnated bone graft is a promising patient-personalized 3D-printed solution for bone defect regeneration, by promoting neighboring host cells recruitment and solid new bone formation. For any figures and tables, please contact the authors directly

A novel EP4 selective agonist (KMN-159) was developed [1] and has been proven that it can act as an osteopromotive factor to repair critical-size femoral bone defects in rats at a dose-dependent manner [2]. Based on its osteopromotive properties, we hypothesized that KMN-159 could also aid in bone formation for spinal fusion. Therefore, the aim of this study was to investigate its spinal fusion effect in a dorsolateral spinal fusion model in rats. This study was performed on 192, 10-week-old male Wistar rats. The rats were randomized into 8 groups (n = 12 per group): 1) SHAM (negative control), 2) MCM (scaffold only), 3) MCM + 20 µg BMP-2 (positive control), 4-8) MCM + 0.2, 2, 20, 200 or 2000 µg KMN-159. A posterolateral intertransverse process spinal fusion at L4 to L5 was performed bilaterally by implanting group dependent scaffolds (see above) or left empty in the SHAM group (protocol no. 25-5131/474/38). Animals were euthanized after 3 weeks and 6 weeks for µCT and biomechanical testing analysis. The results showed that KMN-159 promoted new bone formation in a dose-dependent manner at 3 weeks and 6 weeks as verified by µCT. The biomechanical testing showed that the dose of 20, 200 and 2000 µg KMN-159 groups obtained comparable strength with BMP-2 group, which higher than SHAM, MCM and lower doses of 0.2 and 2 µg KMN-159 groups. In conclusion, KMN-159 could be a potential replacement of BMP-2 as a novel osteopromotive factor for spinal fusion. Acknowledgements: We are grateful to Ulrike Heide, Anna-Maria Placht (assistance with surgeries) as well as Suzanne Manthey & Annett Wenke (histology)

In this work, we combined tissue engineering and gene therapy technologies to develop a therapeutic platform for bone regeneration. We have developed photothermal fibrin-based hydrogels that incorporate degradable CuS nanoparticles (CuSNP) which transduce incident near-infrared (NIR) light into heat. A heat-activated and rapamycin-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in the photothermal hydrogels. In the presence of rapamycin, photoinduced mild hyperthermia induced the release of BMP-2 from the NIR responsive cell constructs. Transcriptome analysis of BMP-2 expressing cells showed a signature of induced genes related to stem cell proliferation and angiogenesis. We next generated 4 mm diameter calvarial defects in the left parietal bone of immunocompetent mice. The defects were filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the gene circuit. After one and eight days, rapamycin was administered intraperitoneally followed by irradiation with an NIR laser. Ten weeks after implantation, the animals were euthanized and samples from the bone defect zone were processed for histological analysis using Masson's trichrome staining and for immunohistochemistry analyses using specific CD31 and CD105 antibodies. Samples from mice that were only administered rapamycin or vehicle or that were only NIR-irradiated showed the persistence of fibrous tissue bridging the defect. In animals that were treated with rapamycin, NIR irradiation of implants resulted in the formation of new mineralized tissue with a high degree of vascularization, thus indicating the therapeutic potential of the approach. Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033)

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future.

Integrin α2β1 is one of the major transmembrane receptors for fibrillary collagen. In native bone we could show that the absence of this protein led to a protective effect against age-related osteoporosis. The objective of this study was to elucidate the effects of integrin α2β1 deficiency on fracture repair and its underlying mechanisms. Standardised femoral fractures were stabilised by an intramedullary nail in 12 week old female C57Bl/6J mice (wild type and integrin α2. -/-. ). After 7, 14 and 28 days mice were sacrificed. Dissected femura were subjected to µCT and histological analyses. To evaluate the biomechanical properties, 28-day-healed femura were tested in a torsional testing device. Masson goldner staining, Alizarin blue, IHC and IF staining were performed on paraffin slices. Blood serum of the animals were measured by ELISA for BMP-2. Primary osteoblasts were analysed by in/on-cell western technology and qRT-PCR. Integrin α2β1 deficient animals showed earlier transition from cartilaginous callus to mineralized callus during fracture repair. The shift from chondrocytes over hypertrophic chondrocytes to bone-forming osteoblasts was accelerated. Collagen production was increased in mutant fracture callus. Serum levels of BMP-2 were increased in healing KO mice. Isolated integrin deficient osteoblast presented an earlier expression and production of active BMP-2 during the differentiation, which led to earlier mineralisation. Biomechanical testing showed no differences between wild-type and mutant bones. Knockout of integrin α2β1 leads to a beneficial outcome for fracture repair. Callus maturation is accelerated, leading to faster recovery, accompanied by an increased generation of extra-cellular matrix material. Biomechanical properties are not diminished by this accelerated healing. The underlying mechanism is driven by an earlier availability of BMP-2, one main effectors for bone development. Local inhibition of integrin α2β1 is therefore a promising target to accelerate fracture repair, especially in patients with retarded healing

Successful application of patient derived cells to engineer vascularized bone grafts is often hampered by low cell numbers and lengthy in vitro expansion. With sound induced morphogenesis (SIM), local cell density enhancement was shown to improve microvasculature formation at lower cell concentration than conventional methods [1]. SIM takes advantage of hydrodynamic forces that act on cells to arrange them within a hydrogel. Following, we are evaluating the potential of cell-hydrogel biografts with high local cell density to improve the therapeutic efficacy in clinical scenarios such as anastomosis or bone formation within non-union fractures. To assess anastomosis, human umbilical vein endothelial cells (HUVEC) and human mesenchymal stromal cells (MSC) were mixed at a 1:1 ratio in PEG-based or Dextran-based hydrogels at a final concentration of 2×10. 6. cells×mL. -1. For ectopic bone formation, MSC were resuspended in PEG-based hydrogels at 2×10. 6. or 5×10. 6. cells×mL. -1. , with or without BMP-2. Cells were assembled into distinct patterns at a frequency of 60 Hz. Four biografts of 4 × 9 mm. 2. were implanted at the back of nude mice (total of 7 animals) and harvested after 2 or 8 weeks. Explants were fixed and imaged as whole constructs or embedded in paraffin for histological analysis. Upon explantation, microscopic evaluation indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks and formed a pre-vascular network. In the second study, ectopic bone formation was more pronounced in areas of higher local cell density based on visual inspection. Ongoing experiments are further characterizing bone formation by micro-CT and histological evaluation. Our results indicate that local cell density enhancement by sound requires a lower initial cell concentration than conventional, static seeding methods to achieve comparable microvasculature structures or local osteogenesis

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions

BMP-1 is the major procollagen-C-peptidase activating, besides fibrillar collagen types I-III, several enzymes and growth factors involved in the generation of extracellular matrix. This study investigated the effect of adding and inhibiting BMP-1 directly post fracture. Standardised femoral fractures were stabilized by an intramedullary nail in 12 week-old female C57Bl/6J mice. We injected either 20 µL recombinant active BMP-1, activity buffer or the BMP-1 specific inhibitor “sizzled”. After 7, 14 and 28 days, mice were sacrificed. Femurs were dissected and paraffin slides were prepared. Callus composition was divided into soft tissue, mineralized and cartilaginous callus. Murine MC3T3 pre-osteoblastic cells were kept in culture adding BMP-1 and sizzled during osteoblastic differentiation. Putative cytotoxicity was determined using MTT-vitality assay. Cell calcification, collagen deposition, and BMP-2 and myostatin protein quantity were characterized. Adding BMP-1 displayed a weak positive effect on the outcome. After 7 days, more mineralised callus was present, meanwhile the cartilaginous callus was apparently remodelled at higher rate. In the case of BMP-1 inhibition, we observed more cartilaginous callus, which may indicate reduced stability. In cell culture, we could observe a high interference with mineralisation capabilities depending on the stage of osteoblastic development when adding BMP-1 or inhibiting it. Addition and inhibition impaired myostatin (anti-osteogen) and BMP-2 (pro-osteogen) expression. Interfering with BMP-1 homeostasis in this early stage of fracture repair seems to have rather negative effects. Inhibition apparently yields lower callus quality while the addition of BMP-1 does not significantly accelerate the healing outcome. Cell culture experiments show that BMP-1 application after 7 days of healing leads to higher collagen output but has no effect on mineralisation. This may suggest that BMP-1 application at a later time-point may lead to more pronounced beneficial effects on fracture repair

Aims

Biofilm-related infection is a major complication that occurs in orthopaedic surgery. Various treatments are available but efficacy to eradicate infections varies significantly. A systematic review was performed to evaluate therapeutic interventions combating biofilm-related infections on in vivo animal models.

Aims

The role of N,N-dimethylformamide (DMF) in diabetes-induced osteoporosis (DM-OS) progression remains unclear. Here, we aimed to explore the effect of DMF on DM-OS development.

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, we report the manufacturing of engineered and devitalized human hypertrophic cartilage (HyC) as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification. Our strategy relies on a customized human mesenchymal line expressing Bone Morphogenetic Protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization and storage, the resulting material exhibited unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy were demonstrated by bioreactor-based production and subsequent orthotopic assessment. Our findings exemplify the broader paradigm of customized ECMs, engineered to activate specific regenerative processes by programming human cell lines as biological factory units

Introduction and Objective. Bone is a tissue which continually regenerates and also having the ability to heal after injuries however, healing of large defects requires intensive surgical treatment. Bioactive glasses are unique materials that can be utilized in both bone and skin regeneration and repair. They are degradable in physiological fluids and have osteoconductive, osteoinductive and osteostimulative properties. Osteoinductive growth factors such as Bone Morphogenetic Proteins (BMP), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF) are well known to stimulate new bone formation and regeneration. Unfortunately, the synthesis of these factors is not cost- effective and, the broad application of growth factors is limited by their poor stability in the scaffolds. Instead, it is wise to incorporate osteoinductive nanomaterials such as graphene nanoplatelets into the structures of synthetic scaffolds. In this study, borate-based 13-93B3 bioactive glass scaffolds were prepared by polymer foam replication method and they were coated with graphene-containing poly (ε-caprolactone) layer to support the bone repair and regeneration. Materials and Methods. Effects of graphene concentration (1, 3, 5, 10 wt%) on the healing of rat segmental femur defects were investigated in vivo using male Sprague–Dawley rats. Fabricated porous bioactive glass scaffolds were coated by graphene- containing polycaprolactone solution using dip coating method. The prepared 0, 1, 3, 5 and 10 wt% graphene nanoparticle-containing PCL-coated composite scaffolds were designated as BG, 1G-P-BG, 3G-P-BG, 5G-P-BG and 10G-P-BG, for each group (n: 4) respectively. Histopathological and immunohistochemical (bone morphogenetic protein, BMP-2; smooth muscle actin, SMA and alkaline phosphatase, ALP) examinations were made after 4 and 8 weeks of implantation. Results. Results showed that after 8-weeks of implantation both cartilage and bone formation were observed in all animal groups. After 4 and 8 weeks of implantation the both osteoblast and osteoclast numbers were significantly higher in the group 4 compared to the control group. Bone formation was significant starting from 1 wt% graphene-coated bioactive glass implanted group and highest amount of bone formation was obtained in group containing 10 wt% graphene (p<0.001). Newly formed vessels expressed this marker and increased vascularization was observed in 8- weeks period compared to the 4-weeks period. In addition, an increase in new vessel formation were observed in graphene-coated scaffold implanted groups compared to the control group. While cartilage tissue was observed in control group, bone formation percentages were significant in graphene-coated scaffold implanted groups. Highest amount of bone formation occurred in group 4 (10 % wt G-C). Conclusions. Additionally, the presence of graphene nanoplatelets enhanced the BMP-2, SMA and ALP levels compared to the bare bioactive glass scaffolds. It was concluded that pristine graphene-coated bioactive glass scaffolds improve osteointegration and bone formation in rat femur defect when compared to bare bioglass scaffolds